Assays to monitor amyloid precursor protein processing

ABSTRACT

The present invention provides DNA constructs, genetically engineered host cells, and methods for identifying inhibitors of amyloid precursor protein (APP) processing. The methods provide for the convenient identification, in a single assay, of inhibitors of β-secretase and γ-secretase as well as other forms of APP processing. The methods rely on fusion proteins of APP and transcription factors in which APP processing releases the transcription factors, allowing the transcription factors to activate transcription of a reporter gene. Inhibitors are identified as substances that block or diminish transcription factor release from the fusion protein, thereby causing a diminution of reporter gene readout.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/360,274, filed Feb. 27, 2002, the contents of which are incorporatedherein by reference in their entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

Not applicable.

REFERENCE TO MICROFICHE APPENDIX

Not applicable.

FIELD OF THE INVENTION

The present invention is directed to the field of Alzheimer's disease.In particular, the present invention provides novel methods ofidentifying substances that are specific inhibitors of various steps inthe processing of amyloid precursor protein.

BACKGROUND OF THE INVENTION

Alzheimer's disease is a common, chronic neurodegenerative disease,characterized by a progressive loss of memory and sometimes severebehavioral abnormalities, as well as an impairment of other cognitivefunctions that often leads to dementia and death. It ranks as the fourthleading cause of death in industrialized societies after heart disease,cancer, and stroke. The incidence of Alzheimer's disease is high, withan estimated 2.5 to 4 million patients affected in the United States andperhaps 17 to 25 million worldwide. Moreover, the number of sufferers isexpected to grow as the population ages.

A characteristic feature of Alzheimer's disease is the presence of largenumbers of insoluble deposits, known as amyloid plaques, in the brainsof those affected. Autopsies have shown that amyloid plaques are foundin the brains of virtually all Alzheimer's patients and that the degreeof amyloid plaque deposition correlates with the degree of dementia(Cummings & Cotman, 1995, Lancet 326:1524-1587). While some opinionholds that amyloid plaques are a late stage by-product of the diseaseprocess, the consensus view is that amyloid plaques are more likely tobe intimately, and perhaps causally, involved in Alzheimer's disease.

A variety of experimental evidence supports this view. For example, Aβ,a primary component of amyloid plaques, is toxic to neurons in cultureand transgenic mice that overproduce Aβ in their brains show significantdeposition of Aβ into amyloid plaques as well as significant neuronaltoxicity (Yankner, 1990, Science 250:279-282; Mattson et al., 1992, J.Neurosci. 12:379-389; Games et al., 1995, Nature 373:523-527; LaFerla etal., 1995, Nature Genetics 9:21-29). Mutations in the APP gene, leadingto increased Aβ production, have been linked to heritable forms ofAlzheimer's disease (Goate et al., 1991, Nature 349:704-706;Chartier-Harlan et al., 1991, Nature 353:844-846; Murrel et al.,1991,Science 254:97-99; Mullan et al., 1992, Nature Genetics 1:345-347).Presenilin-1 (PS 1) and presenilin-2 (PS2) related familial early-onsetAlzheimer's disease (FAD) shows disproportionately increased productionof Aβ1-42, the 42 amino acid isoform of Aβ, as opposed to Aβ1-40, the 40amino acid isoform (Scheuner et al, 1996, Nature Medicine 2:864-870).The longer isoform of Aβ is more prone to aggregation than the shorterisoform (Jarrett et al, 1993, Biochemistry 32:4693-4697). Injection ofthe insoluble, fibrillar form of Aβ into monkey brains results in thedevelopment of pathology (neuronal destruction, tau phosphorylation,microglial proliferation) that closely mimics Alzheimer's disease inhumans (Geula et al., 1998, Nature Medicine 4:827-831). See Selkoe,1994, J. Neuropathol. Exp. Neurol. 53:438-447 for a review of theevidence that amyloid plaques have a central role in Alzheimer'sdisease.

Aβ, a 39-43 amino acid peptide derived by proteolytic cleavage of theamyloid precursor protein (APP), is the major component of amyloidplaques (Glenner & Wong, 1984, Biochem. Biophys. Res. Comm.120:885-890). APP is actually a family of polypeptides produced byalternative splicing from a single gene. Major forms of APP are known asAPP₆₉₅, APP₇₅₁, and APP₇₇₀, with the subscripts referring to the numberof amino acids in each splice variant (Ponte et al., 1988, Nature331:525-527; Tanzi et al., 1988, Nature 331:528-530; Kitaguchi et al.,1988, Nature 331:530-532). APP is membrane bound and undergoesproteolytic cleavage by at least two pathways. In one pathway, cleavageby an enzyme known as α-secretase occurs while APP is still in thetrans-Golgi secretory compartment (Kuentzel et al., 1993, Biochem. J.295:367-378). This cleavage by α-secretase occurs within the Aβ portionof APP, thus precluding the formation of Aβ. In another proteolyticpathway, cleavage of the Met₅₉₆-Asp₅₉₇ bond (numbered according to the695 amino acid protein) by an enzyme known as β-secretase occurs. Thiscleavage by β-secretase generates the N-terminus of Aβ. The C-terminusis formed by cleavage by a second enzyme known as γ-secretase. TheC-terminus is actually a heterogeneous collection of cleavage sitesrather than a single site since γ-secretase activity occurs over a shortstretch of APP amino acids rather than at a single peptide bond.Peptides of 40 or 42 amino acids in length (Aβ1-40 and Aβ11-42,respectively) predominate among the C-termini generated by γ-secretase.Aβ1-42 is more prone to aggregation than Aβ1-40, is the major componentof amyloid plaque (Jarrett et al., 1993, Biochemistry 32:4693-4697; Kuoet al., 1996, J. Biol. Chem. 271:4077-4081), and its production isclosely associated with the development of Alzheimer's disease (Sinha &Lieberburg, 1999, Proc. Natl. Acad. Sci. USA 96:11049-11053). The bondcleaved by γ-secretase appears to be situated within the transmembranedomain of APP. It is unclear as to whether the C-termini of Aβ1-40 andAβ1-42 are generated by a single γ-secretase protease with sloppyspecificity or by two distinct proteases. For a review that discussesAPP and its processing, see Selkoe, 1998, Trends Cell. Biol. 8:447-453.

Much interest has focused on the possibility of inhibiting thedevelopment of amyloid plaques as a means of preventing or amelioratingthe symptoms of Alzheimer's disease. To that end, a promising strategyis to inhibit the activity of β- and γ-secretase, the two enzymes thattogether are responsible for producing Aβ. This strategy is attractivebecause, if the formation of amyloid plaques as a result of thedeposition of Aβ is a cause of Alzheimer's disease, inhibiting theactivity of one or both of the two secretases would intervene in thedisease process at an early stage, before late-stage events such asinflammation or apoptosis occur. Such early stage intervention isexpected to be particularly beneficial (see, e.g., Citron, 2000,Molecular Medicine Today 6:392-397).

To that end, various assays have been developed that are directed to theidentification of compounds that may interfere with the production of Aβor its deposition into amyloid plaques. U.S. Pat. No. 5,441,870 isdirected to methods of monitoring the processing of APP by detecting theproduction of amino terminal fragments of APP. U.S. Pat. No. 5,605,811is directed to methods of identifying inhibitors of the production ofamino terminal fragments of APP. U.S. Pat. No. 5,593,846 is directed tomethods of detecting soluble Aβ by the use of binding substances such asantibodies. Esler et al., 1997, Nature Biotechnology 15:258-263described an assay that monitored the deposition of Aβ from solutiononto a synthetic analogue of an amyloid plaque. The assay was suitablefor identifying compounds that could inhibit the deposition of Aβ.However, this assay is not suitable for identifying substances, such asinhibitors of β- or γ-secretase, that would prevent the formation of Aβ.

Various groups have cloned and sequenced cDNA encoding a protein that isbelieved to be β-secretase (Vassar et al., 1999, Science 286:735-741;Hussain et al., 1999, Mol. Cell. Neurosci. 14:419-427; Yan et al., 1999,Nature 402:533-537; Sinha et al., 1999, Nature 402:537-540; Lin et al.,2000, Proc. Natl. Acad. Sci. USA 97:1456-1460) but the identity ofγ-secretase has been more elusive. A pair of proteins known aspresenilin-1 and presenilin-2 are viewed as possible candidates (Selkoe& Wolfe, 2000, Proc. Natl. Acad. Sci. USA 97:5690-5692).

Presenilin-1 (PS1) and presenilin-2 (PS2) are polytopic membraneproteins that are involved in γ-secretase-mediated processing of APP.The most common cause of familial early-onset Alzheimer's disease is theautosomal dominant inheritance of assorted mutations in the PS1 gene(Sherrington et al., 1995, Nature 375:754-760). These PS1 mutations leadto increased production of Aβ1-42 (Scheuner et al., 1996, NatureMedicine 2:864-870; Duff et al., 1996, Nature 383:710-713; Borchelt etal., 1996, Neuron 17:1005-1013). Similarly, certain mutations in PS2cause familial early-onset Alzheimer's disease and increased generationof Aβ42 (Levy-Lahad et al., 1995, Science 269:970-973). Culturedisolated neurons from PS1-deficient mice exhibit reducedγ-secretase-mediated cleavage of APP (De Strooper et al., 1998, Nature391:387-390). It was suggested that PS1 might influence trafficking ofAPP and/or γ-secretase or it might play a more direct role inproteolytic cleavage of APP. Directed mutagenesis of two conservedtransmembrane-situated aspartates in PS1 was shown to inactivateγ-secretase activity in cellular assays, suggesting that PS1 is either arequired diaspartyl cofactor for γ-secretase or is itself γ-secretase(Wolfe et al., 1999, Nature 398:513-517). Moreover, Li et al., 2000,Nature 405:689-694 made photoactivatable derivatives of a highlyspecific and potent aspartyl protease transition state analog inhibitorand found that the inhibitor selectively labeled presenilin fragments.

Co-immunoprecipitation experiments have shown that PS1 and PS2 interactdirectly with the immature forms of APP in the endoplasmic reticulumwhere the disease-associated amyloid Aβ1-42 peptide is probablygenerated (Xia et al., 1997 Proc. Natl. Acad. Sci. USA 94:8208-8213;Weidemann et al., 1997, Nat. Med. 3:328-332). Knock-out of PS1 activitygreatly diminishes γ-secretase cleavage of APP (De Strooper et al.,1998, Nature 391:387-390). PS1 knock-outs do not exhibit total lack ofγ-secretase activity but knock-out of both PS1 and PS2 activity doesresult in a total loss of γ-secretase activity (Herreman et al., 2000,Nat. Cell. Biol. 2:461-462; Zhang et al., 2000, Nat. Cell Biol.2:463-465), suggesting that PS2 has a similar function to PS1 in theprocessing of APP.

Karlström et al., (Journal of Biological Chemistry papers in press,published on Dec. 13, 2001 as Manuscript C100649200) describes an assaydesigned specifically to identify inhibitors of γ-secretase cleavage ofAPP. The authors inserted the GAL4 DNA binding domain fused to the VP16transactivation domain into C99, a portion of APP containing the 99carboxy-terminal amino acids. This fragment of APP contains theγ-secretase cleavage site but lacks the β-secretase cleavage site.Transaction of a UAS reporter plasmid by GAL4-VP16 confirmed cleavage ofthe Gal4-VP16/C99 substrate by γ-secretase only. Thus, the assay iscapable of detecting γ-secretase inhibitors but not inhibitors ofβ-secretase or other modulators of APP processing requiring theN-terminal domain of APP.

Cao & Stühoff, 2001, Science 293:115-120 described work in which theGAL4 and LexA DNA binding domains were inserted into APP to demonstratethe potential of the cleaved C-terminus of APP for transcriptionalco-activation. In this article, a transcriptional factor was not fusedto APP and no attempt was made to develop an assay for theidentification of APP processing inhibitors.

Sisodia, 1992, Proc. Natl. Acad. Sci. USA 89:6975-6979 described variouschanges in the amino acid sequence of APP in the region of theα-secretase cleavage site and the effect of those changes on cleavage byα-secretase. A change of K to V at position 612 of the 695 amino acidversion of APP led to reduced cleavage by α-secretase.

U.S. Pat. No. 6,333,167 B1 discloses an assay involving DNA constructsencoding portions of membrane proteins containing sites that aresusceptible to cleavage by proteases that are fused to transcriptionalrepressors. Such constructs are introduced into cells that contain areporter gene under the control of a promoter that is sensitive to therepressor. In the absence of an inhibitor of the protease, the fusionprotein is cleaved by the protease, releasing a membraneprotein/repressor fusion protein that translocates to the nucleus andrepresses transcription from the reporter gene. In the presence of aninhibitor of the protease, the membrane protein/repressor fusion proteinis not released and thus cannot repress transcription from the reporter.An increase in reporter expression can therefore be used as a readoutfor the presence of an inhibitor.

SUMMARY OF THE INVENTION

The present invention is directed to methods of identifying inhibitorsof the processing of amyloid precursor protein (APP) that are capable ofidentifying inhibitors of a number of steps of such processing. Unlikeprior methods, the methods of the present invention can be used toscreen for inhibitors of β-secretase cleavage, γ-secretase cleavage, APPextracellular signaling, or APP cytoplasmic signaling in a single assay.

The methods employ a recombinant eukaryotic cell that is capable ofprocessing APP. The cell has been engineered to express a fusion proteinthat contains amino acid sequences encompassing both the β-secretasecleavage site of APP and the γ-secretase cleavage site. The fusionprotein also contains a transcription factor fused in frame to the APPsequences.

When the recombinant cell is further engineered to contain a reportergene, in which transcription of the reporter gene is driven by aregulatory DNA sequence that is inactive in the absence of thetranscription factor but active in the transcription factor's presence,a system useful for screening for APP processing inhibitors is provided.Since the recombinant cell has been selected so as to be capable ofprocessing APP, the fusion protein will be processed, releasing thetranscription factor and activating transcription of the reporter gene.The reporter gene has been preselected so that activation of thereporter gene leads to a detectable phenotype.

The system is utilized by exposing the recombinant cell to substancesthat are to be tested for the ability to inhibit APP processing. Thosesubstances that are actually inhibitors of APP processing will causediminished processing of the fusion protein, leading to smaller amountsof the transcription factor being released. This leads to lesstranscription of the reporter gene. This results in a decrease in thephenotypic effect of the reporter gene that can be observed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-G shows a schematic diagram of several APP/transcription factorfusion constructs.

FIG. 2A-B shows the DNA sequence (SEQ ID NO:1) of the fusion proteinAPP(1-651)SW, K612V-TATexonI(M1L) APP (664-695).

FIG. 3 shows the amino acid sequence (SEQ ID NO:2) of the fusion proteinAPP(1-651)SW, K612V-TATexonI(M1L) APP (664-695) with the variousdifferent regions of the fusion protein demarcated. 1=amino acids 1-651of APP; 2=region of β-secretase cleavage; 3=K612V mutation; 4=region ofγ-secretase cleavage; 5=linker; 6=TAT exon I; 7=linker; 8=amino acids664-695 of APP.

FIG. 4A-B shows the DNA sequence (SEQ ID NO:3) of the fusion proteinAPP(1-651)wt, K612V-TATexonI(M1L) APP (664-695).

FIG. 5 shows the amino acid sequence (SEQ ID NO:4) of the fusion proteinAPP(1-651)wt, K612V-TATexonI(M1L) APP (664-695) with the variousdifferent regions of the fusion protein demarcated. 1=amino acids 1-651of APP; 2=region of β-secretase cleavage; 3=K612V mutation; 4=region ofγ-secretase cleavage; 5=linker; 6=TAT exon I; 7=linker; 8=amino acids664-695 of APP.

FIG. 6A-C shows the DNA sequence (SEQ ID NO:5) of the fusion proteinAPP(1-651)SW, K612V, GAL4-VP16(delMet) APP (664-695).

FIG. 7 shows the amino acid sequence (SEQ ID NO:6) of the fusion proteinAPP(1-651)SW, K612V, GAL4-VP16(delMet) APP (664-695) with the variousdifferent regions of the fusion protein demarcated. 1=amino acids 1-651of APP; 2=region of β-secretase cleavage; 3=K612V mutation; 4=region ofγ-secretase cleavage; 5=linker; 6=GAL4-VP16; 7=linker; 8=amino acids664-695 of APP.

FIG. 8A-C shows the DNA sequence (SEQ ID NO:7) of the fusion proteinAPP(1-651)wt, K612V, GAL4-VP16(del Met) APP (664-695).

FIG. 9 shows the amino acid sequence (SEQ ID NO:8) of the fusion proteinAPP(1-651)wt, K612V, GAL4-VP16(del Met) APP (664-695) with the variousdifferent regions of the fusion protein demarcated. 1=amino acids 1-651of APP; 2=region of β-secretase cleavage; 3=K612V mutation; 4=region ofγ-secretase cleavage; 5=linker; 6=GAL4-VP16; 7=linker; 8=amino acids664-695 of APP.

FIG. 10A-B shows the DNA sequence (SEQ ID NO:9) of the fusion proteinAPP(1-651)SW, TATexonI(M1L) APP (664-695).

FIG. 11 shows the amino acid sequence (SEQ ID NO:10) of the fusionprotein APP(1-651)SW, TATexonI(M1L) APP (664-695) with the variousdifferent regions of the fusion protein demarcated. 1=amino acids 1-651of APP; 2=region of β-secretase cleavage; 3=wild-type K at position 612;4=region of γ-secretase cleavage; 5=linker; 6=TAT exon I; 7=linker;8=amino acids 664-695 of APP.

FIG. 12A-B shows the DNA sequence (SEQ ID NO:11) of the fusion proteinAPP(1-651)wt, TATexonI(M1L) APP (664-695).

FIG. 13 shows the amino acid sequence (SEQ ID NO:12) of the fusionprotein APP(1-651)wt, TATexonI(M1L) APP (664-695) with the variousdifferent regions of the fusion protein demarcated. 1=amino acids 1-651of APP; 2=region of 13-secretase cleavage; 3=wild-type K at position612; 4=region of γ-secretase cleavage; 5=linker; 6=TAT exon I; 7=linker;8=amino acids 664-695 of APP.

FIG. 14A-C shows the DNA sequence (SEQ ID NO:13) of the fusion proteinAPP(1-651)SW, GAL4-VP16(delMet) APP (664-695).

FIG. 15 shows the amino acid sequence (SEQ ID NO:14) of the fusionprotein APP(1-651)SW, GAL4-VP16(delMet) APP (664-695) with the variousdifferent regions of the fusion protein demarcated. 1=amino acids 1-651of APP; 2=region of β-secretase cleavage; 3=wild-type K at position 612;4=region of γ-secretase cleavage; 5=linker; 6=GAL4-VP16; 7=linker;8=amino acids 664-695 of APP.

FIG. 16A-C shows the DNA sequence (SEQ ID NO:15) of the fusion proteinAPP(1-651)wt, GAL4-VP16(delMet) APP (664-695).

FIG. 17 shows the amino acid sequence (SEQ ID NO:16) of the fusionprotein APP(1-651)wt, GAL4-VP16(delMet) APP (664-695) with the variousdifferent regions of the fusion protein demarcated. 1=amino acids 1-651of APP; 2=region of β-secretase cleavage; 3=wild-type K at position 612;4=region of γ-secretase cleavage; 5=linker; 6=GAL4-4VP16; 7=linker;8=amino acids 664-695 of APP.

FIG. 18A-B shows the cDNA sequence (SEQ ID NO:17) and FIG. 18C shows theamino acid sequence (SEQ ID NO:18) of the 695 amino acid splice variantof wild-type Alzheimer's precursor protein (APP). See GenBank accessionno. Y00264 and Kang et al., 1987, Nature 325:733-736.

FIG. 19 shows data from an embodiment in which the assay of the presentinvention was used to identify both a β-secretase inhibitor and aγ-secretase inhibitor. See Example 3 for details.

FIG. 20 shows a schematic diagram of pCR2.1 Gal4-VP16.

FIG. 21A shows a schematic diagram of pRBR121. FIG. 21B shows aschematic diagram of pcDNA3.1 zeo (+) APP(1-651)SW, K612V-TATexonI(M1L)APP (664-695).

FIG. 22A shows a schematic diagram of pRBR186. FIG. 22B shows aschematic diagram of the viral plasmid pNL4-3. FIG. 22C shows aschematic diagram of pcDNA3.1 zeo (+) APP(1-651)SW, K612V-TATexonI(M1L)APP (664-695) with additional details as compared to FIG. 21B, whichshows the same plasmid.

FIG. 23 shows a schematic diagram of pRSV Kan/Neo res.

FIG. 24 shows a schematic diagram of pUCd5TAT.

FIG. 25A shows a schematic diagram of pMM321. FIG. 25B-D shows thenucleotide sequence of pMM321. The upper strand is SEQ ID NO:19. Thelower strand (SEQ ID NO:20) is the reverse complement of SEQ ID NO:19.

FIG. 26A shows a schematic diagram of the expression vector pcDNA3.1 zeo(+)APP(1-651)SW, K612V-(M1L)TATexonI. This expression vector directs theexpression of a fusion protein containing the first 651 amino acids ofAPP with the Swedish version of the β-secretase cleavage site and theK612V mutation fused to the first exon of HIV1 TAT. The methionine atposition 1 of TAT has been changed to leucine. FIG. 26B-G shows thenucleotide sequence of pcDNA3.1 zeo (+)APP(1-651)SW,K612V-(M1L)TATexonI. The upper strand is SEQ ID NO:21. The lower strand(SEQ ID NO:22) is the reverse complement of SEQ ID NO:21.

FIG. 27A-B shows a schematic diagram depicting general features of thepresent invention. FIG. 27A: The vertical bar represents a fusionprotein with APP sequences represented as unfilled or lightly shadedportions of the bar. The lightly shaded portion represents Aβ. “BACE”indicates the β-secretase cleavage site. The dark shaded portionrepresents the transcription factor fused between APP sequences. Thehorizontal bar represents a membrane in which the uncleaved fusionprotein is embedded, e.g., the endoplasmic reticulum. FIG. 27B: Thetranscription factor (plus small amounts of APP), having been releasedfrom the fusion protein and thus the membrane by APP processing, isshown in the nucleus binding to and activating the regulatory DNAsequence (“Transcription Factor Response Element”) that controls theexpression of the reporter gene.

FIG. 28A-B shows the DNA sequence (SEQ ID NO:23) of the fusion proteinAPP(1-651)NFEV, K612V-TATexonI(M1L) APP (664-695).

FIG. 29 shows the amino acid sequence of a fusion protein(APP(1-651)NFEV, K612V-TATexonI(M1L) APP (664-695)) (SEQ ID NO:24)containing the sequence NFEV at the β-secretase cleavage site(underlined at 2). The other portions of the fusion protein areindicated as follows: 1=amino acids 1-651 of APP; 2=region ofβ-secretase cleavage; 3=K612V mutation; 4=region of γ-secretasecleavage; 5=linker; 6=TAT exon I; 7=linker; 8=amino acids 664-695 ofAPP.

FIG. 30A-C shows the DNA sequence (SEQ ID NO:25) of the fusion proteinAPP(1-651)NFEV, K612V, GAL4-VP16(delMet) APP (664-695).

FIG. 31 shows the amino acid sequence of a fusion protein(APP(1-651)NFEV, K612V, GAL4-VP16(delMet) APP (664-695)) (SEQ ID NO:26)containing the sequence NFEV at the β-secretase cleavage site(underlined at 2). The other portions of the fusion protein areindicated as follows: 1=amino acids 1-651 of APP; 2=region ofβ-secretase cleavage; 3=K612V mutation; 4=region of γ-secretasecleavage; 5=linker; 6=GAL4-VP16; 7=linker; 8=amino acids 664-695 of APP.

FIG. 32A shows a schematic diagram of pcDNA3.1 zeo (+), a eukaryoticexpression vector that is suitable for use in the present invention.FIG. 32B-F shows the nucleotide sequence of pcDNA3.1 zeo (+). The upperstrand is SEQ ID NO:27. The lower strand (SEQ ID NO:28) is the reversecomplement of SEQ ID NO:27.

FIG. 33 shows data from an embodiment of the present invention utilizinga β-galactosidase reporter gene in which the assay of the presentinvention was used to identify both a β-secretase inhibitor and aγ-secretase inhibitor. See Example 8 for details.

FIG. 34 shows data from an embodiment of the present invention in whicha fusion protein having a wild-type β-secretase cleavage site and afusion protein having a Swedish β-secretase cleavage site are compared.See Example 9 for details.

FIG. 35A-B shows the DNA sequence (SEQ ID NO:29) of the fusion proteinAPP(1-651)NFEV, TATexonI(M1L) APP (664-695).

FIG. 36 shows the amino acid sequence of a fusion protein(APP(1-651)NFEV, TATexonI(M1L) APP (664-695)) (SEQ ID NO:30) containingthe sequence NFEV at the β-secretase cleavage site (underlined at 2) anda wild-type K at position 612 (underlined at 3). The other portions ofthe fusion protein are indicated as follows: 1=amino acids 1-651 of APP;2=region of β-secretase cleavage; 3=wild-type K; 4=region of γ-secretasecleavage; 5=linker; 6=TAT exon I; 7=linker; 8=amino acids 664-695 ofAPP.

FIG. 37A-C shows the DNA sequence (SEQ ID NO:3 1) of the fusion proteinAPP(1-651)NFEV, GAL4-VP16(delMet) APP (664-695).

FIG. 38 shows the amino acid sequence of a fusion protein(APP(1-651)NFEV, GAL4-VP16(delMet) APP (664-695)) (SEQ ID NO:32)containing the sequence NFEV at the β-secretase cleavage site(underlined at 2) and a wild-type K at position 612 (underlined at 3).The other portions of the fusion protein are indicated as follows:1=amino acids 1-651 of APP; 2=region of β-secretase cleavage;3=wild-type K; 4=region of γ-secretase cleavage; 5=linker; 6=GAL4-VP16;7=linker; 8=amino acids 664-695 of APP.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of this invention:

A “fusion protein” is a protein that contains at least two polypeptideregions and, optionally, a linking peptide to operatively link the twopolypeptides into one continuous polypeptide. The at least twopolypeptide regions in a fusion protein are derived from differentsources, and therefore a fusion protein comprises two polypeptideregions not normally joined together in nature.

A “linking sequence (or linker peptide)” contains one or more amino acidresidues joined in peptide bonds. A linking sequence serves to join twopolypeptide regions of differing origins in a fusion protein via apeptide bond between the linking sequence and each of the polypeptideregions.

Typically, a fusion protein is synthesized as a continuous polypeptidein a recombinant host cell which contains an expression vectorcomprising a nucleotide sequence encoding the fusion protein where thedifferent regions of the fusion protein are fused in frame on eitherside of a linker peptide's coding sequence. The chimeric coding sequence(encoding the fusion protein) is operatively linked to expressioncontrol sequences (generally provided by the expression vector) that arefunctional in the recombinant host cell.

“Reporter gene,” as used in the present invention, does not mean a DNAsequence present on the chromosome of a cell, generally possessingintrons, as is often meant by the word “gene” in the art. Rather“reporter gene” means any DNA sequence encoding a protein or polypeptidethat can give rise to a signal that can be detected or measured.“Reporter gene” does not mean a portion of the amino acid sequence ofAPP. “Reporter gene” will usually mean a DNA sequence, generally a cDNAsequence (although in some cases a reporter gene may have introns) thatencodes a protein or polypeptide that is commonly used in the art toprovide a measurable phenotype that can be distinguished over backgroundsignals.

A “nuclear localization signal (NLS)” is a region of a polypeptide whichtargets the polypeptide to the nucleus of the cell. One such NLS is thatfrom the SV40 large T antigen. See, e.g., U.S. Pat. No. 5,589,392;Kalderon et al., 1984, Cell 39:499-509. The minimum region of the SV40large T antigen with NLS activity is Pro-Lys-Lys-Lys-Arg-Lys-Val (SEQ IDNO:22). See also U.S. Pat. No. 5,776,689.

“Substances” that are screened in the present invention can be anysubstances that are generally screened in the pharmaceutical industryduring the drug development process. For example, substances may be lowmolecular weight organic compounds (e.g., having a molecular weight ofless than about 2,000 daltons and preferably less than about 1,000daltons), RNA, DNA, antibodies, peptides, or proteins. Substances areoften tested in the methods of the present invention as largecollections of substances, e.g. libraries of low molecular weightorganic compounds, peptides, or natural products.

The conditions under which substances are employed in the methodsdescribed herein are conditions that are typically used in the art forthe study of protein-ligand interactions or enzyme inhibition studies:e.g., salt conditions such as those represented by such commonly usedbuffers as PBS or in tissue culture media; a temperature of about 4° C.to about 55° C.; incubation times of from several seconds to severalhours or even up to 24 or 48 hours. Screening for the identification ofenzyme-specific inhibitors is a well-known procedure in thepharmaceutical arts and the numerous conditions under which suchscreening has been done are available in the literature to guide thepractitioner of he present invention.

A “conservative amino acid substitution” refers to the replacement ofone amino acid residue by another, chemically similar, amino acidresidue. Examples of such conservative substitutions are: substitutionof one hydrophobic residue (isoleucine, leucine, valine, or methionine)for another; substitution of one polar residue for another polar residueof the same charge (e.g., arginine for lysine; glutamic acid foraspartic acid); substitution of one aromatic amino acid (tryptophan,tyrosine, or phenylalanine) for another.

“Transfection” refers to any of the methods known in the art forintroducing DNA into a cell, e.g., calcium phosphate or calcium chloridemediated transfection, electroporation, infection with a retroviralvector.

The present invention relates to the discovery of an assay system thatpermits the simultaneous screening for inhibitors of several types ofamyloid precursor protein (APP) processing or signaling (e.g.,β-secretase cleavage, γ-secretase cleavage, APP extracellular signaling,APP cytoplasmic signaling). In a preferred embodiment, this screening isaccomplished without the concomitant identification of inhibitors ofα-secretase. The assay system is carried out in a single type of cell,using a single type of assay readout. Inhibitors discovered by means ofthe present invention are expected to be useful in the treatment ofAlzheimer's disease since these inhibitors are likely to be capable ofinterfering with the production of Aβ.

Previous assays for identifying inhibitors of APP processing havefocussed specifically on inhibition of either β-secretase or γ-secretaseactivity, or on inhibition of some other single aspect of Aβ production.In contrast, the assays described herein are directed to inhibition ofAPP processing in general. Substances identified through these assaysmay target β-secretase, γ-secretase, modulators of β-secretase orγ-secretase activity, or even an as-yet-undiscovered ligand interactionwith APP. In certain embodiments, these assays will also be free of thepotentially misleading or obscuring effects of α-secretase activity. Inaddition, unlike other assays currently in use, these assays arehomogeneous assays; i.e., they require no cumbersome or time-consumingsteps such as column chromatography separations, immunoprecipitations,washing steps, etc. Therefore, the assays are very well adapted to ahigh throughput screening format.

In the present invention, novel recombinant DNA molecules areconstructed in which nucleotide sequences encoding at least a portion ofthe luminal (i.e., N-terminal to the transmembrane region) andtransmembrane regions of APP are fused to nucleotide sequences encodinga transcription factor. In a preferred embodiment, the APP contains anα-secretase cleavage site that has been altered to reduce or eliminateα-secretase cleavage. This allows the assays of the present invention toavoid identifying inhibitors of α-secretase and permits the moreefficient detection of β-secretase inhibitors since α-secretase andβ-secretase compete for APP cleavage. The recombinant DNA molecules maybe transfected, along with a reporter gene, into a cell line thatprocesses APP into Aβ, and stable clones may be generated.Alternatively, the recombinant DNA molecules and reporter plasmid may beutilized in transient transfections.

Upon expression in cells, the APP/transcription factor fusion proteinlocalizes to a non-nuclear membrane of the cell (e.g., the endoplasmicreticulum) due to the presence of the APP sequences in the fusionprotein. In a manner similar to cleavage of APP, the fusion protein willthen be cleaved, first by β-secretase and then by γ-secretase.γ-secretase cleavage releases the transcription factor from the membranein which the APP/transcription factor fusion protein had been embedded,after which the transcription factor translocates to the nucleus andstimulates transcription of the reporter gene. Assuming no α-secretasecleavage, cleavage by both β-secretase and γ-secretase is required forrelease of the transcription factor and transactivation of the reportergene in this assay since γ-secretase cleavage of APP is dependent on ashort luminal domain, such as that generated by β- or α-secretasecleavage. Detection of a signal from the reporter gene product will thusserve as evidence of APP processing. In particular, since activation ofthe reporter gene requires both β-secretase and γ-secretase cleavage,the assay is capable of identifying inhibitors of both or either ofthese proteases.

FIG. 27 is a schematic diagram depicting general features of the assay.The vertical bar in FIG. 27A represents the fusion protein; thehorizontal bar represents the non-nuclear membrane in which the fusionprotein is embedded before processing. FIG. 27B shows how thetranscription factor portion of the fusion protein (with small amountsof the APP portion flanking it) has moved to the nucleus followingrelease from the fusion protein by APP processing. In the nucleus, thetranscription factor is shown binding to a regulatory DNA sequence(“Transcription Factor Response Element”) and activating transcriptionof the reporter gene.

The recombinant DNA molecules encoding the APP/transcription factorfusion protein and the reporter gene can be used to develop novelhomogenous cell-based assays for the identification and assessment ofinhibitors of APP processing which will be readily amenable to highthroughput technology.

In one embodiment, the recombinant DNA molecules used in this inventioncomprise sequences encoding the amino terminal 651 amino acids of the695 amino acid version of APP (Kang et al., 1987, Nature 325:733-736),including all the sequences necessary for the production of Aβ, as wellas the C-terminal 32 amino acids of APP. The transcription factor isplaced between the N-terminal and C-terminal portions of APP. The APPsequence may include a modification to increase the amount ofβ-secretase cleavage of the fusion protein. This modification involvesmutating the K at position 612 of the α-secretase cleavage site to a V(K612V). Since α-secretase and β-secretase compete for APP cleavage,reducing or eliminating APP cleavage by α-secretase results in increasedβ-secretase cleavage, and allows the assay to detect β-secretaseinhibitors more readily. In addition, the β-secretase cleavage sitewithin APP (KM↓DA) (SEQ ID NO:34) may be modified, e.g., to that of anaturally occurring mutation (termed the “Swedish” mutation or NL↓DA)(SEQ ID NO:38) which has been shown to enhance β-secretase cleavagesix-fold in cultured cells. Another possible modification is to replacethe (KM↓DA) (SEQ ID NO:34) wild-type β-secretase cleavage site with thesequence (NF↓EV) (SEQ ID NO:40). The presence of NFEV in an amino acidsequence has been shown to enhance β-secretase cleavage by an evenlarger amount than the Swedish sequence. See U.S. Provisional PatentApplication Ser. No. 60/292,591 and U.S. Provisional Patent ApplicationSer. No. 60/316,115, the disclosures of which are incorporated herein,in their entirety.

In a preferred embodiment, HIV-1 TAT exon I has been fused betweensequences encoding the first 651 amino acids of APP₆₉₅ and the last 32amino acids of APP₆₉₅ (APP-TAT-APPct32). Co-transfection of anexpression vector comprising this construct with a reporter gene plasmidcontaining an HIV-1 LTR promoter that controls the transcription of areporter gene leads to enhanced expression of the reporter gene. Othertranscription factors that could be fused to APP₁₋₆₅₁ include Gal4-VP16,the entire Gal4 protein, BIV TAT, HIV-2 TAT, SIV TAT, LexA-VP16, EBVZta, Papillomavimus E2, or tissue or species specific homodimeric bHLHtranscription factors capable of activating transcription throughspecific DNA response elements, such as E12, E47, or Twist. The use ofGAL4, BIV, HIV-2, or SIV TAT may be useful if it is desired to reducethe potency of the transactivator, thus reducing any backgroundtransactivation caused by non-specific cleavage of the fusion protein.To further reduce the potential for transactivation by TAT in theabsence of β-secretase and γ-secretase cleavage, the TAT portion of thefusion protein may be altered to remove the N-terminal methionine andthus eliminate the possibility of aberrant translation of TAT throughany potential internal ribosomal entry sites.

In some circumstances, high level expression of TAT has been found to betoxic to cells. Thus, when TAT is the transcription factor fused to APPin the methods of the present invention, it may be advantageous toutilize transient transfection with low amounts of the expression vectorencoding the APP/TAT fusion protein. A set of preliminary experiments inwhich various amounts of the vector are transfected, in order to titrateacceptable levels of TAT, is recommended.

The reporter gene used will depend in large part upon the transcriptionfactor fused to APP. The promoter used to drive the reporter gene willbe LTR for TAT-based APP fusion proteins, or UAS (6×) forGAL4-VP16-based APP fusion proteins. In a particular embodiment, an LTRdriving EGFP (enhanced green fluorescent protein, a brighter variant ofGFP made by Aurora Biosciences, San Diego, Calif.) has been used toobserve processing of an APP/TAT fusion protein. Under certainconditions, it may be desirable to use a less stable reporter, such asdsEGFP (a destabilized variant of EGFP made by Aurora Biosciences, SanDiego, Calif. and marketed by Clontech, Palo Alto, Calif.) or a morepotent reporter, such as β-lactamase. Alternatively, a stable HeLa cellline expressing LTR-β-galactosidase can be used. If the exquisitesensitivity of β-lactamase makes it less than optimal for a particularpurpose, the LTR-β-galactosidase cell line may be exploited for thisassay. Finally, under some circumstances Gal4-VP16 may prove to beoptimal relative to TAT to reduce any inherent background problemsassociated with using the weakly but constitutively active LTR in thereporter plasmid, in which case the reporter plasmid could beUAS(6×)-β-lactamase (Aurora Biosciences, San Diego, Calif.).

A variety of cells are suitable for use in the methods of the presentinvention. Particularly preferred are eukaryotic, especially mammalian,cell lines. In particular embodiments, the cells are selected from thegroup consisting of: L cells L-M(TK⁻) (ATCC CCL 1.3), L cells L-M (ATCCCCL 1.2), HEK293 (ATCC CRL 1573), HEK293T, Raji (ATCC CCL 86), CV-1(ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1(ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCCCCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), T24 (ATCC HTB-4),PC12 cells, Jurkat cells, H4 cells (ATCC HTB-148), and MRC-5 (ATCC CCL171).

To make the assay more amenable for ultra-high throughput screening, anon-adherent cell line, such as Jurkat, can be used.

Generally, the assays of the present invention employ cells thatnaturally express β-secretase and γ-secretase. However, it is possibleto practice the invention in cells that lack the expression of one, orboth, of these enzymes. In such cases, β-secretase and γ-secretaseactivity can be provided by the recombinant expression of these enzymesin the cells.

In one embodiment, the present invention provides a recombinant cell,preferably a eukaryotic cell, even more preferably a mammalian cell, andmost preferably a human cell, where the cell expresses a fusion proteinof APP and a transcription factor and the cell contains a reporter genethat can be activated by the transcription factor. The fusion proteincomprises a portion of APP where that portion includes the regions ofthe β-secretase and γ-secretase cleavage sites fused to a transcriptionfactor. The region of APP including the β-secretase and γ-secretasecleavage sites can be, e.g., a portion of APP that includes amino acids589-651 of the 695 amino acid version of APP. This region is shownbelow.         ↓ EEISEVKM DAEFRHDSGYEVHHQKLVFFAEDVGS (SEQ ID NO:33)              ↓  ↓ NKGAIIGLMVGGVVIA TVIVITLVMLKKK

The β-secretase cleavage site is shown at position 596-597    ↓ (KM DA).(SEQ ID NO:34)

Two predominant cleavage sites of γ-secretase are shown at positions636-637 and 638-639     ↓  ↓ (GVV IA TV). (SEQ ID NO > 35)

The fusion protein will be anchored in the membrane by the APP sequencesshown above. The N-terminal portion of APP must include at least theβ-secretase cleavage site, and possibly several amino-acids N-terminalto the β-secretase cleavage site to make the assay sensitive to bothβ-secretase and γ-secretase inhibitors. In many cases, the APP sequenceswill include sequences further N-terminal than those shown above,including the signal sequence at the N-terminus of APP. In cases, wherethe APP signal sequence is not used, another signal sequence may beincorporated in the fusion protein. Such other signal sequences areknown in the art.

In a related embodiment, the present invention provides a recombinantcell, preferably a eukaryotic cell, even more preferably a mammaliancell, and most preferably a human cell, where the above-described APPportion of the fusion protein contains a K612V mutation. The APP portionof this embodiment is shown below.         ↓ EEISEVKMDAEFRHDSGYEVHHQVLVFFAEDVGS (SEQ ID NO:36)               ↓  ↓NKGAIIGLMVGGVVIA TVIVITLVMLKKK

The β-secretase cleavage site is shown at position 596-597    ↓ (KM DA).(SEQ ID NO:34)

Two predominant cleavage sites of γ-secretase are shown at positions636-637 and 638-639     ↓  ↓ (GVV IA TV). (SEQ ID NO:35)The underlined V at position 612 shows the change in sequence in thepresent invention from the wild-type K to the mutant V, which changeprovides for reduced cleavage by α-secretase.

In a related embodiment, the present invention provides a recombinantcell, preferably a eukaryotic cell, even more preferably a mammaliancell, and most preferably a human cell, where the above-described APPportion of the fusion protein contains the Swedish version of theβ-secretase cleavage site as well as a K612V mutation. The APP portionof this embodiment is shown below.         ↓ EEISEVNLDAEFRHDSGYEVHHQVLVFFAEDVGS (SEQ ID NO:37)               ↓  ↓NKGAIIGLMVGGVVIA TVIVITLVMLKKK

The β-secretase cleavage site is shown at position 596-597    ↓ (NL DA).(SEQ ID NO:38)

Two predominant cleavage sites of γ-secretase are shown at positions636-637 and 638-639     ↓  ↓ (GVV IA TV). (SEQ ID NO:35)The underlined V at position 612 shows the change in sequence in thepresent invention from the wild-type K to the mutant V, which changeprovides for reduced cleavage by α-secretase.

In a related embodiment, the present invention provides a recombinantcell, preferably a eukaryotic cell, even more preferably a mammaliancell, and most preferably a human cell, where the above-described APPportion of the fusion protein contains the NFEV version of theβ-secretase cleavage site as well as a K612V mutation. The APP portionof this embodiment is shown below.         ↓ EEISEVNFEVEFRHDSGYEVHHQVLVFFAEDVGS (SEQ ID NO:39)               ↓  ↓NKGAIIGLMVGGVVIA TVIVITLVMLKKK

The β-secretase cleavage site is shown at position 596-597    ↓ (NF EV).(SEQ ID NO:40)

Two predominant cleavage sites of γ-secretase are shown at positions636-637 and 638-639     ↓  ↓ (GVV IA TV). (SEQ ID NO:35)The underlined V at position 612 shows the change in sequence in thepresent invention from the wild-type K to the mutant V, which changeprovides for reduced cleavage by α-secretase.

The presence of both β-secretase and γ-secretase cleavage sites in thefusion proteins permits the assays of the present invention to detectinhibitors of both β-secretase and γ-secretase.

The recombinant host cells of the present invention can be furtherengineered to comprise a reporter gene construct. The reporter geneconstruct contains a reporter gene in operable linkage with a regulatoryDNA sequence that confers on the reporter gene the property of beingregulated by the transcription factor of the fusion protein. Thisregulation is such that expression of the reporter gene is low or absentwithout binding of the transcription factor to the regulatory DNAsequence but, when the transcription factor is released from the fusionprotein by APP processing, the transcription factor can move into thenucleus of the cell and bind to the regulatory DNA sequence, therebyactivating transcription from the reporter gene.

Reporter genes desirably give rise to gene products which can bedetected or quantitated, either in terms of amount of proteinsynthesized, enzymatic activity, fluorescence, luminescence, or someother phenotype. Suitable reporter gene products include fireflyluciferase (de Wet et al., 1987, Mol. Cell. Biol. 7:725-737) orbacterial luciferase (Englebrecht et al., 1985, Science 227:1345-1347;Baldwin et al., 1984, Biochem. 23:3663-3667), β-lactamase,β-glucuronidase, β-galactosidase, green fluorescent proteins, enhancedgreen fluorescent protein, destabilized enhanced green fluorescentprotein, red fluorescent protein, yellow fluorescent protein, cyanfluorescent protein, destabilized yellow fluorescent protein,destabilized cyan fluorescent protein, aequorin, chloramphenicol acetyltransferase (Alton & Vapnek, 1979, Nature 282:864-869), rat liveralkaline phosphatase (Toh et al., 1989, Eur. J. Biochem. 182:231-237),human placental secreted alkaline phosphatase (Cullen & Mallim, 1992,Meth. Enzymol. 216:362-368), and horseradish peroxidase, among others.

A preferred reporter gene is green fluorescent protein (GFP) or amodified GFP. Wild-type GFP has long been used in the art. Starting fromgreen fluorescent protein, many modified versions have been derived withaltered or enhanced spectral properties as compared with wild-type GFP.See, e.g., U.S. Pat. No. 5,625,048; International Patent Publication WO97/28261; International Patent Publication WO 96/23810. Useful are themodified GFPs W1B and TOPAZ, available commercially from AuroraBiosciences Corp., San Diego, Calif. W1B contains the following changesfrom the wild-type GFP sequence: F64L, S65T, Y66W, N146I, M153T, andV163A (see Table 1, page 519, of Tsien, 1998, Ann. Rev. Biochem.67:509-544). TOPAZ contains the following changes from the wild-type GFPsequence: S65G, V68L, S72A, and T203Y (see Table 1, page 519, of Tsien,1998, Ann. Rev. Biochem. 67:509-544). Wild-type nucleotide and aminoacid sequences of GFP are shown in FIG. 1 and SEQ ID NO:1 ofInternational Patent Publication WO 97/28261; in FIG. 1 of Tsien, 1998,Ann. Rev. Biochem. 67:509-544; and in Prasher et al., 1992, Gene111:229-233.

When expressing GFPs in mammalian cells, it may be advantageous toconstruct versions of the GFPs having altered codons that conform tothose codons preferred by mammalian cells (Zolotukhin et al., J. Virol.1996, 70:4646-46754; Yang et al., 1996, Nucl. Acids Res. 24:4592-4593).Another way of improving GFP expression in mammalian cells is to providean optimal ribosome binding site by the use of an additional codonimmediately after the starting methionine (Crameri et al., 1996, NatureBiotechnology 14:315-319).

Transcription factors that are useful in the present invention arepreferably those transcription factors that are not naturally expressedin the recombinant host cells. This is so the regulatory DNA sequence isnot activated absent APP processing and release of the transcriptionfactor from the fusion protein. Preferably, the transcription factorcontains, or is engineered to contain, a nuclear localization signal.This is so that, after release, the transcription factor will move intothe nucleus of the genetically modified host cells where it can bind to,and activate, the regulatory DNA sequence, leading to expression of thereporter gene. Transcription factors, as used in the present invention,do not include proteins that, after release from a fusion protein andtranslocation into the nucleus, repress transcription from a reportergene.

Among the transcription factors that are useful in the present inventionare: HIV1 TAT (in particular exon I of HIV1 TAT), Gal4-VP16, the entireGal4 protein, BIV TAT, HIV-2 TAT, SIV TAT, LexA-VP16, EBV Zta,Papillomavirus E2, or one of the bHLM homodimeric transcription factors,E12, E47, or Twist.

Expression vectors are generally used to express the fusion protein inthe recombinant cells. An expression vector contains recombinant nucleicacid encoding a polypeptide (e.g., an APP/transcription factor fusionprotein) along with regulatory elements for proper transcription andprocessing. Generally, the regulatory elements that are present in anexpression vector include a transcriptional promoter, a ribosome bindingsite, a transcriptional terminator, and a polyadenylation signal. Otherelements may include an origin of replication for autonomous replicationin a host cell, a selectable marker, a limited number of usefulrestriction enzyme sites, and a potential for high copy number.

A variety of expression vectors are known in the art and can be used inthe present invention. Commercially available expression vectors whichare suitable include, but are not limited to, pMC1neo (Stratagene), pSG5(Stratagene), pcDNAI and pcDNAIamp, pcDNA3, pcDNA3.1, pCR3.1(Invitrogen, San Diego, Calif.), EBO-pSV2-neo (ATCC 37593), pBPV-1(8-2)(ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199),pRSVneo (ATCC 37198), pCI.neo (Promega), pTRE (Clontech, Palo Alto,Calif.), pV1Jneo, pIRESneo (Clontech, Palo Alto, Calif.), pCEP4(Invitrogen, San Diego, Calif.), pSC11, and pSV2-dhfr (ATCC 37146). Thechoice of vector will depend upon the cell type in which it is desiredto express the APP/transcription factor fusion protein, as well as onthe level of expression desired, and the like.

The expression vectors can be used to transiently express or stablyexpress the fusion protein. The transient expression or stableexpression of transfected DNA is well known in the art. See, e.g.,Ausubel et al., 1995, “Introduction of DNA into mammalian cells,” inCurrent Protocols in Molecular Biology, sections 9.5.1-9.5.6 (John Wiley& Sons, Inc.).

The recombinant host cells of the present invention are useful inmethods of screening substances for the ability to inhibit APPprocessing. In one embodiment, the methods of the present inventioncomprise adding a candidate substance to a recombinant host cellcomprising an APP/transcription factor fusion protein and a reportergene and comparing the level of expression of the reporter gene proteinin the presence and absence of the candidate substance, wherein thelevel of expression of the reporter gene protein is lower when thecandidate substance inhibits processing of the APP/transcription factorfusion protein such that the transcription factor is not released, or isreleased in a lower amount, than in the absence of the substance.

The level of expression of the reporter gene protein is generally notmeasured directly. Rather, an indirect method is used. For example,fluorescence given off by the reporter gene protein may be detected ormeasured as, e.g., when the reporter gene product is a green fluorescentprotein; or, some enzymatic activity of the reporter gene product may bedetected or measured, e.g., when the reporter gene product isβ-lactamase.

The candidate substance may be of any form suitable for entry into thecytoplasm of the recombinant cell or for contact with the cell'scytoplasmic membrane. Under appropriate conditions, the candidatesubstance may be allowed to freely diffuse into the cell, or thedelivery of the substance may be facilitated by techniques andsubstances which enhance cell permeability, a wide variety of which areknown in the art. Methods for increasing cell permeability include,without limitation, the use of organic solvents such asdimethylsulfoxide, liposomes, application of electrical current, andphysical means such as substance-coated teflon pellets.

The present invention provides a method of identifying a substance thatinhibits APP processing comprising:

(a) providing a recombinant eukaryotic cell which:

-   -   (i) expresses a fusion protein comprising amino acids 589-651 of        APP₆₉₅ and a transcription factor where the transcription factor        is fused in frame to the carboxyl terminus of amino acids        589-651 of APP₆₉₅; and    -   (ii) comprises a reporter gene operably linked to a regulatory        DNA sequence which is capable of being activated by the        transcription factor;

(b) measuring the level of reporter gene product in the cell in theabsence of the substance;

(c) adding the substance to the cell and measuring the level of reportergene product in the cell in the presence of the substance;

where a decrease in the level of reporter gene product in the presenceas compared to the absence of the substance indicates that the substanceinhibits APP processing.

The manner in which the level of the reporter gene product is measuredwill be determined by the nature of the reporter gene and, often, thecharacteristics of the host cell. For example, if the reporter geneproduct itself is fluorescent, as for example, when a green fluorescentprotein is the reporter gene product, fluorescence from the cell can bemeasured directly. When the reporter gene product has enzymaticactivity, for example, when the reporter gene product is β-lactamase,known methods of measuring that enzymatic activity can be used.

For the sake of clarity, the above method is described in terms of “a”cell. In actual practice, the method will generally be carried on alarge number of cells at one time. For example, the method will often becarried out in a well of a tissue culture plate, where, depending on thenumber of wells in the plate (and thus their size), there can be up tohundreds, thousands, or even several million cells. The step of “addingthe substance to the cell” is generally carried out by simply adding thesubstance to the tissue culture medium in which the cells are present.After the substance is added to the cell, the cell and the substance areincubated for a period of time sufficient for the substance to inhibitAPP processing, if the substance is actually an inhibitor of APPprocessing. This period is usually from about 15 minutes to 48 hours,but may be somewhat more in unusual cases.

A convenient way of carrying out the method is to grow a population ofthe recombinant eukaryotic cells and then split the population into aportion that will be exposed to the substance and a portion that willnot be exposed to the substance.

The recombinant eukaryotic cell is generally produced by transfection ofan expression vector encoding the fusion protein and by transfection ofa plasmid containing the reporter gene.

One skilled in the art would recognize that what is sought in terms of“a decrease in the level of reporter gene product in the presence ascompared to the absence of the substance” is a non-trivial decrease. Forexample, if in the method described above there is found a 1% decrease,this would not indicate that the substance is an inhibitor of APPprocessing. Rather, one skilled in the art would attribute such a smalldecrease to normal experimental variance. What is looked for is asignificant decrease. For the purposes of this invention, a significantdecrease fulfills the usual requirements for a statistically validmeasurement of a biological signal. For example, depending upon thedetails of the embodiment of the invention, a significant decrease mightbe a decrease of at least 10%, preferably at least 20%, more preferablyat least 50%, and most preferably at least 90%.

In particular embodiments, amino acids 589-651 of APP₆₉₅ contain a K612Vmutation.

In particular embodiments, the cell is a mammalian cell. In particularembodiments, the cell is a human cell.

In particular embodiments, the method is used to screen a library ofmore than 1,000 substances. In other embodiments, the method is used toscreen a library of more than 50,000 substances at a rate of more than1,000 substances per 24 hours.

In particular embodiments, the fusion protein comprises a portion of APPthat is selected from the group consisting of: amino acids 1-651 ofAPP₆₉₅, amino acids 50-651 of APP₆₉₅, amino acids 100-651 of APP₆₉₅,amino acids 150-651 of APP₆₉₅, amino acids 200-651 of APP₆₉₅, aminoacids 250-651 of APP₆₉₅, amino acids 300-651 of APP₆₉₅, amino acids350-651 of APP₆₉₅, amino acids 400-651 of APP₆₉₅, amino acids 450-651 ofAPP₆₉₅, amino acids 500-651 of APP₆₉₅, and amino acids 550-651 ofAPP₆₉₅.

In related embodiments, the fusion protein does not comprise all ofamino acids 589-651 of APP₆₉₅. Rather, the fusion protein comprisesslightly fewer amino acids from APP. For example, the fusion proteinmight comprise slightly fewer amino acids of the β-secretase cleavagesite: e.g., amino acids 590-651 of APP₆₉₅. Or the fusion protein mightcomprise slightly fewer amino acids of the γ-secretase cleavage site:amino acids 589-650 of APP₆₉₅; amino acids 589-649 of APP₆₉₅; aminoacids 589-648 of APP₆₉₅; or amino acids 589-647. The fusion protein mayeven comprise slightly fewer amino acids from both ends, e.g., aminoacids 590-647 of APP₆₉₅. What is important is that the portion of APPincluded in the fusion protein contains both the β-secretase cleavagesite and the γ-secretase cleavage site.

In particular embodiments, the transcription factor is selected from thegroup consisting of: HIV-1 TAT, Gal4-VP16, the entire Gal4 protein,LexA-VP16, EBV Zta, Papillomavirus E2, one of the bHLH homodimerictranscription factors, including E12, E47, or Twist, or BIV TAT, BIV-2TAT, or SIV TAT. A particular version of HIV-1 TAT suitable for use inthe present invention is HIV-1 TAT exon I.

Fusion proteins suitable for use in the present invention can beselected from the group consisting of: APP(1-651)SW, K612V-TATexonI(M1L)APP (664-695) (SEQ ID NO:2); APP(1-651)wt, K612V-TATexonI(M1L) APP(664-695) (SEQ ID NO:4); APP(1-651)SW, K612V, Gal4-VP16(1L) APP(664-695) (SEQ ID NO:6); APP(1-651)wt, K612V, Gal4-VP16(M1L) APP(664-695) (SEQ ID NO:8); APP(1-651)SW, TATexonI(M1L) APP (664-695) (SEQID NO:10); APP(1-651)wt, TATexonI(M1L) APP (664-695) (SEQ ID NO:12);APP(1-651)SW, Gal4-VP16(M1L) APP (664-695) (SEQ ID NO:14); APP(1-651)wt,Gal4-VP16(M1L) APP (664-695) (SEQ ID NO:16); APP(1-65 1)NFEV,K612V-TATexonI(M1L) APP (664-695) (SEQ ID NO:23); and APP(1-651)NFEV,K612V, GAL4-VP16(M1L) APP (664-695) (SEQ ID NO:25).

In some embodiments of the present invention, the amino acid sequencescontributed to the fusion protein by the transcription factor constitutethe carboxy terminal amino acid sequences of the fusion protein. Inother embodiments, the transcription factor has other sequences fused toits carboxy terminus, as in the examples herein where amino acids664-695 of APP₆₉₅ are fused to the carboxy terminus of the transcriptionfactor and therefore constitute the carboxy terminal amino acidsequences of the fusion protein. Other portions of APP (e.g., aminoacids 652-695 of APP₆₉₅) could be used instead of amino acids 664-695 ofAPP₆₉₅. In fact, it should be possible to extend the carboxy terminus ofthe transcription factor with almost any amino acid sequences, providingsuch sequences do not interfere with the ability of the transcriptionfactor to move into the nucleus and activate transcription of thereporter gene once the transcription factor has been released from thefusion protein by the action of γ-secretase.

The present invention includes a method of identifying a substance thatinhibits APP processing comprising:

(a) providing a recombinant eukaryotic cell which:

-   -   (i) expresses a fusion protein comprising an amino acid sequence        from APP that is capable of being cleaved by both β-secretase        and γ-secretase and a transcription factor where the        transcription factor is fused in frame to the amino acid        sequence from APP; and    -   (ii) comprises a reporter gene operably linked to a regulatory        DNA sequence which capable of being activated by the        transcription factor;

(b) measuring the level of reporter gene product in the cell in theabsence of the substance;

(c) adding the compound to the cell and measuring the level of reportergene product in the cell in the presence of the substance;

where a decrease in the level of reporter gene product in the presenceas compared to the absence of the substance indicates that the substanceinhibits APP processing.

In particular embodiments, the amino acid sequence from APP comprises:

589-651 of APP₆₉₅;

590-651 of APP₆₉₅;

589-650 of APP₆₉₅;

590-650 of APP₆₉₅;

589-649 of APP₆₉₅;

590-649 of APP₆₉₅;

589-648 of APP₆₉₅;

590-648 of APP₆₉₅;

589-647 of APP₆₉₅; or

590-647 of APP₆₉₅.

In related embodiments, the amino acid sequence from APP contains theamino acid sequence NLDA (SEQ ID NO:38) at the β-secretase cleavage siteinstead of the wild-type sequence KMDA (SEQ ID NO:34).

In related embodiments, the amino acid sequence from APP contains theamino acid sequence NFEV (SEQ ID NO:40) at the β-secretase cleavage siteinstead of the wild-type sequence KMDA (SEQ ID NO:34).

The portion of the fusion protein that is derived from APP may containmutations that are known in the art. Of particular interest aremutations that result in an increased proportion of Aβ being made in theform of Aβ1-42 rather than Aβ1-40. Such mutations are disclosed in thefollowing publications (numbering is from the 770 amino acid version ofAPP):

-   Swedish (K670N, M671L): Mullan et al., 1992, Nature Genet.    1:345-347.-   Flemish (A692G): Hendriks et al., 1992, Nature Genet. 1:218-221;    Cras et al., 1998, Acta Neuropathol. (Berlin) 96:253-260.-   Dutch (E693Q): Levy et al., 1990, Science 248:1124-1126.-   Arctic (E693G): Nilsberth et al., 2001, Nature Neuroscience 4:    887-893.-   Austrian (T714I): Kumar-Singh et al., 2000, Hum. Mol. Genet.    9:2589-2598.-   French (V715M): Ancolio et al., 1999, Proc. Natl. Acad. Sci. (USA)    96:4119-4124.-   Florida (I716V): Eckman et al., 1997, Hum. Mol. Genet. 6:2087-2089.-   V717F: Murrell et al., 1991, Science 254:97-99.-   V717G: Chartier-Harlin et al., 1991, Nature 353:844-846.-   London (V717I): Goate et al., 1991, Nature 349:704-706.-   L723P: Kwok et al., 2000, Ann. Neurol. 47:249-253.-   I716F (also called I45F, referring to the position relative to the    β-secretase cleavage site): This mutation in APP changes processing    of Aβ almost exclusively to Aβ1-42.-   Lichtenthaler et al., 1999, Proc. Natl. Acad. Sci. (USA)    96:3053-3058.

As with many proteins, it may be possible to modify many of the aminoacids of the fusion proteins described above and still retainsubstantially the same biological activity in terms of APP processing asfor the original fusion protein. Thus, the present invention includesmodified fusion proteins which have amino acid deletions, additions, orsubstitutions but that still retain substantially the same propertieswith respect to APP processing as the fusion proteins described herein.It is generally accepted that single amino acid substitutions do notusually alter the biological activity of a protein (see, e.g., MolecularBiology of the Gene, Watson et al., 1987, Fourth Ed., TheBenjamin/Cummings Publishing Co., Inc., page 226; and Cunningham &Wells, 1989, Science 244:1081-1085). Accordingly, the present inventionincludes fusion proteins where one amino acid substitution has been madein the fusion proteins described herein where the fusion proteins stillretain substantially the same properties with respect to APP processingas the fusion proteins described herein. The present invention alsoincludes fusion proteins where two or more amino acid substitutions havebeen made in the fusion proteins described herein where the fusionproteins still retain substantially the same properties with respect toAPP processing as the fusion proteins described herein. In particular,the present invention includes embodiments where the substitutions areconservative substitutions.

With the exception of FIG. 18, the nucleotide and amino acid sequencesof APP disclosed herein contain a minor difference compared to APPsequences that are usually reported in the literature. For the sequencesdisclosed herein with such a difference, the nucleotide at position 367is an A rather than a G, as in most published APP sequences. This changeresults in a conservative substitution in the corresponding APP aminoacid sequence. Thus, the amino acid sequences disclosed herein with sucha difference have an I rather than a V at position 123. This differencedoes not affect the properties of the fusion proteins for the purposesof the present invention. Therefore, fusion proteins having the APPsequence reported in the literature with an G at nucleotide position 367and a V at amino acid position 123 and the fusion proteins disclosedherein with an A at nucleotide position 367 and an I at amino acidposition 123 are to be considered equivalents for the purposes of thepresent invention.

The Gal-VP16 sequences disclosed herein contain two changes from theusual published sequences. There is T to C change at nucleotide position2131 that causes a S to P change at amino acid position 712; there is Ato C change at nucleotide position 2301 that does not change the aminoacid sequence. It is expected that Gal-VP16 proteins containing theusual sequences reported in the literature will also be suitable for usein the present invention.

The methods of the present invention can be used to screen libraries ofsubstances or other sources of substances to identify substances thatare inhibitors of β-secretase or γ-secretase. Such identified inhibitorysubstances can serve as “leads” for the development of pharmaceuticalsthat can be used to treat patients having Alzheimer's disease or in aprophylactic manner to prevent or delay the development of Alzheimer'sdisease. Such leads can be further developed into pharmaceuticals by,for example, subjecting the leads to sequential modifications, molecularmodeling, and other routine procedures employed in the pharmaceuticalindustry. The inhibitors of APP processing identified by the presentinvention may also be tested in animal models of Alzheimer's diseasesuch as the various transgenic mouse models that are known in the art.

Although a wide variety of substances can be screened by the methods ofthe present invention, preferred substances for screening are librariesof small molecule compounds. Small molecule compounds are preferredbecause they are more readily absorbed after oral administration, havefewer potential antigenic determinants, and are more likely to cross theblood/brain barrier than larger molecules such as nucleic acids orproteins.

Once identified by the methods of the present invention, the candidatesmall molecule compounds may then be produced in quantities sufficientfor pharmaceutical testing and formulated in a pharmaceuticallyacceptable carrier (see, e.g., Remington's Pharmaceutical Sciences,Gennaro, A., ed., Mack Publishing, 1990, for suitable methods). Thecandidate compounds may be administered to cell lines relevant toAlzheimer's disease, animal models of Alzheimer's disease, orAlzheimer's disease patients.

The numbering of the amino acids in APP used herein is based on the 695amino acid version of APP described in Kang et al., 1987, Nature325:733-736. There are two other major versions of APP, having 751 aminoacids and 770 amino acids (see, Ponte et al., 1988, Nature 331:525-527for the 751 amino acid version and Kitaguchi et al., 1988, Nature331:530-532 for the 770 amino acid version). One skilled in the art willunderstand how to translate the numbering used herein, based on the 695amino acid version of APP, into the corresponding numbering for otherversions of APP. For example, some of the APP/transcription factorfusion proteins of the present invention contain the K612V mutation,based on the numbering of the 695 amino acid version. This mutationwould correspond to a K668V mutation in the 751 amino acid version and aK687V mutation in the 770 amino acid version.

Therefore, when a “K612V” mutation is referred to herein, it will beunderstood that such reference also includes a K668V mutation of the 751amino acid version of APP as well as a K687V mutation of the 770 aminoacid version of APP.

Similarly, the portion of APP referred to as APP₁₋₆₅₁ herein, based onthe 695 amino acid version, will be understood to mean also APP₁₋₇₀₇ ofthe 751 amino acid version and APP₁₋₇₂₆ of the 770 amino acid version.

If desired, inhibitors that are identified by the methods of the presentinvention can be further tested to determine which step in APPprocessing they affect. Assays that are known to be specific for thevarious steps of APP processing can be used for this purpose. Forexample, the assay of Karlström et al., (Journal of Biological Chemistrypapers in press, published on Dec. 13, 2001 as Manuscript C100649200) isonly capable of detecting inhibitors of γ-secretase and cannot alsodetect inhibitors of other steps of APP processing such as, e.g.,inhibitors of β-secretase. If an inhibitor identified by the methods ofthe present invention is found to also be an inhibitor when tested inthe assay of Karlström et al., then that inhibitor is at least aγ-secretase inhibitor. It is still possible that that inhibitor couldinhibit other steps in APP processing as well. Further tests known inthe art can determine this.

The present invention may be modified so as to provide methods ofdetermining at which step of APP processing a known inhibitor of APPprocessing exerts its effect. The known inhibitor may be one that hasbeen identified by the methods of the present invention or by some othermethod. The modification to the present invention consists in mutatingthe β-secretase site in a fusion protein so that β-secretase cleavagecan no longer occur at the site or occurs at a very much reduced level.Providing that the fusion protein contains a cleavable α-secretase site,the fusion protein can still be used in the methods of the presentinvention. However, this fusion protein (with a mutated β-secretasesite) can no longer detect β-secretase inhibitors. Therefore, if theknown APP processing inhibitor still functions as an APP processinginhibitor in this modified version of the invention, then the knowninhibitor cannot be a β-secretase site inhibitor but instead must exertits effect downstream of β-secretase.

Suitable mutations of the β-secretase site include the following. Allthe sequences are for amino acid positions 594-598 of APP₆₉₅. VNFAV (SEQID NO:41): This mutation shows decreased β-secretase cleavage relativeto the wild type, KMDA (SEQ ID NO:34), sequence. VKVDA (SEQ ID NO:42):Vassar et al., 1999, Science 286:735-741. This mutant was tested invitro only, but purified β-secretase failed to cleave a 30-amino acidpeptide containing this sequence.

WKMDA (SEQ ID NO:43), VKADA (SEQ ID NO:44), VKKDA (SEQ D) NO:45), VKEDA(SEQ ID NO:46), VKIDA (SEQ ID NO:47), VKMIA (SEQ ID NO:48), VKMNA (SEQID NO:49), VKUEA (SEQ ID NO:50), VKMDE (SEQ ID NO:51), VKMDK (SEQ IDNO:52): Citron et al., 1995. Neuron 14:661-670. These mutationsdecreased Aβ production 4-20× relative to p3 production in culturedcells.

Fusion proteins can be constructed by use of the polymerase chainreaction (PCR) to amplify desired portions of APP and transcriptionfactors, which can be then be cloned into expression vectors by methodswell known in the art. Primers for PCR will generally include a smallpart of the APP or transcription factor as well as convenient cloningsites and/or linker peptide sequences. The PCR primers can be used toamplify the desired APP or transcription factor fragments from sourcessuch as previously cloned APP or transcription factors, cDNA libraries,or genomic libraries. The amplified APP and transcription factorsequences can be cloned into suitable expression vectors. Methods of PCRand cloning are well known in the art and can be found in standardreference materials such as those listed below.

Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are known and commonly employed by those skilledin the art. A number of standard techniques are described in Sambrook etal. (1989) Molecular Cloning, Second Edition, Cold Spring HarborLaboratory, Plainview, N.Y.; Maniatis et al. (1982) Molecular Cloning,Cold Spring Harbor Laboratory, Plainview, N. Y.; Wu (ed.) (1993) Meth.Enzymol. 218, Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wu et al.(eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.)Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Oldand Primrose (1981) Principles of Gene Manipulation, University ofCalifornia Press, Berkeley; Schleif and Wensink (1982) Practical Methodsin Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRLPress, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic AcidHybridization, IRL Press, Oxford, UK; Setlow and Hollaender (1979)Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press,New York, and Ausubel et al. (1992) Current Protocols in MolecularBiology, Greene/Wiley, New York, N.Y.

PCR reactions can be carried out with a variety of thermostable enzymesincluding but not limited to AmpliTaq, AmpliTaq Gold, or Ventpolymerase. For AmpliTaq, reactions can be carried out in 10 mM Tris-Cl,pH 8.3, 2.0 mM MgCl₂, 200 μM of each dNTP, 50 mM KCl, 0.2 μM of eachprimer, 10 ng of DNA template, 0.05 units/μl of AmpliTaq. The reactionsare heated at 95° C. for 3 minutes and then cycled 35 times usingsuitable cycling parameters, including, but not limited to, 95° C., 20seconds, 62° C., 20 seconds, 72° C., 3 minutes. In addition to theseconditions, a variety of suitable PCR protocols can be found in PCRPrimer, A Laboratory Manual, edited by C. W. Dieffenbach and G. S.Dveksler, 1995, Cold Spring Harbor Laboratory Press; or PCR Protocols: AGuide to Methods and Applications, Michael et al., eds., 1990, AcademicPress.

It is desirable to sequence the DNA encoding the fusion proteins, or atleast the junction regions of the various portions (APP, transcriptionfactor, linkers) of the fusion protein in order to verify that thedesired portions have in fact been obtained, joined properly, and thatno unexpected changes have been introduced into the sequences by the PCRreactions.

Suitable PCR primers for amplification of DNA sequences for use in thepresent invention can be readily designed by those of skill in the art.Examples of such primers are shown below.

-   5′-GGA GAG GAT ATC ATG GAG CCA GTA GAT CC-3′ (SEQ ID NO:53) can be    used to amplify the 5′ portion of HIV-1 TAT exon I.-   5′-TAC ATG GCG GCC GCC TAC TTA CTG CTT TG-3′ (SEQ ID NO:54) can be    used to amplify the 3′ portion of HIV-1 TAT exon I.-   5′-GGA TGT GAT ATC TTT CTT CTT CAG CAT CAC CAA GG-3′ (SEQ ID NO:55)    can be used to amplify the 3′ portion of DNA encoding amino acids    1-651 of APP, i.e., the transmembrane region of APP.

The following non-limiting examples are presented to better illustratethe invention.

EXAMPLE 1 Transfection of pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI With pMM321

The following example demonstrated that an APP/TAT fusion construct willtransactivate a reporter gene in which the HIV1 LTR regulatory DNAsequence controls the expression of enhanced green fluorescent protein(EGFP). The following also serves as an example of the kind ofpreliminary routine variations of fusion protein levels and inhibitorlevels that may be advantageous to test in the practice of the presentinvention. Such routine variations are often helpful in validating theassays before a large scale screening project is undertaken.

The APP/TAT fusion construct is referred to as “pcDNA3.1 zeo (+)APP(1-651)SW, K612V-(M1L)TATexonI” (see FIG. 26) and contains the HIV1TAT exon 1 fused just after the transmembrane domain of APP. Thisconstruct is also shown in outline form in FIG. 1B. “pMM321” refers to areporter gene plasmid consisting of the HIV1 LTR driving thetranscription of enhanced green fluorescent protein (see FIG. 25). As apositive control for TAT expression, a construct in which TAT was underthe control of a strong, constitutive promoter ( referred to as“pUCd5TAT”; see FIG. 24) was used.

Methods:

-   1. Day 1: Pass HEK 293T cells into 2×6 well dishes at 1×10⁵    cells/well.-   2. Day 2: Transfect cells with 9 μL Fugene and 0.125 μg pMM321 and    various amounts of pcDNA3.1 zeo (+) APP(1-651)SW,    K612V-(M1L)TATexonI.    Plate 1

1. 5 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI

2. 2.5 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI

3. 1.25 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI

4. 0.625 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI

5. 0.312 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI

6. 0.156 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI

Plate 2:

1. 0.08 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI

2. 0.04 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI

3. no pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI

4. 0.625 pUCd5TAT

5. 0.625 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI and 1 μgpMM321

6. 1 μg pMM321

Six hours post-transfection, green cells were only observed in plate 2,#5.

-   3. Day 3: The fluorescence intensity of the transfected cells was    observed and recorded.-   4. Day 4: The fluorescence intensity of transfected cells was    observed and recorded.    Results:

Co-transfection with pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonIincreased GFP expression in the cells.

Day 3:

5 μg—no green cells

2.5 μg—no green cells (too much DNA for these two transfections?)

1.25 μg—many bright and dim green cells (see photographs and figure inancillary data)

0.625 μg—bright and dim green cells but fewer than at 1.25 μg

0.312 μg—no difference obvious between 0.625 μg and 0.312 μg

0.156 μg—very few green cells

0.08 μg—very few green cells

0.04 μg—very few green cells

no pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI—extremely few (ifany) green cells

0.625 μg pUCd5TAT—cells were extremely bright, not necessarily more innumber than with pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI

0.625 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI +1 μgpMM321—many bright and dim green cells.

1 μg pMM321 many-fold fewer green cells, some bright, most dim.

Day 3—changed media (saved 1 mL conditioned media from wells Plate 1-3,4, 5, 6; Plate 2-1, 2, 3, 5, 6). Added fresh media with 10 μM ofL-685,458 (a potent, cell permeable γ-secretase inhibitor) to wellsPlate 1-3, 4, 5, 6; Plate 2-3, 4, 5, 6. Waited 48 hours to observe lossof fluorescence since GFP is so stable.

After 48 hours, all wells appeared brighter than at 24 hour time point.This does not necessarily mean that the inhibitor was ineffective, orthat the assay did not work, since there were no controls run where theinhibitor was not added. However, it does suggest that under theseconditions it may be preferable to add the inhibitor at the time oftransfection to shut down γ-secretase as soon as possible and avoidrelease of TAT and induction of GFP.

EXAMPLE 2 Transfection of APP(1-651)SW, K612V-(MIL)TATexonI Into HEK293Tand H4 Cells Accompanied By Inhibition of γ-secretase Activity WithL-685,458

The following example demonstrates the operation of the invention inHEK293T cells and H4 cells and shows inhibition of APP processing (andthus TAT release) by treatment with a known γ-secretase inhibitor.“pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI,” “pMM321,” and“pUCd5TAT” are the same as in Example 1. H4 cells (ATCC HTB-148) are aneuronal cell line.

Methods:

-   Day 1: Plated out 2×6 well plates of HEK293T cells and 2×6 well    plates of H4 cells at 1×10⁵ cells/well.-   Day 2: Transfected cells with 2 μg total DNA—pcDNA3.1 zeo (+)    APP(1-651)SW, K612V-(M1L)TATexonI and carrier (a pET-IN plasmid).    Plate 1:

1,2: 1 μg pMM321+1 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI

3,4: 1 μg pMM321+0.1 μg pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI+0.9 μg carrier

5: 1 μg pMM321+1 μg carrier (added too much carrier to this well in H4cells)

6: 1 μg pMM321+0.1 μg pUCd5TAT+0.9 μg carrier

Plate 2:

1,2: 0.1 μg pMM321+1 μg pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI+0.9 μg carrier

3,4: 0.1 μg pMM321+0.1 μg pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI+1.8 μg carrier (added too 0.5×carrier to this mix in293T cells)

5: 0.1 μg pMM321+1.9 μg carrier

6. 0.1 μg pMM321+0.1 μg pUCd5TAT+1.8 μg carrier

Transfections for HEK293T cells: 9 μL Fugene/well. Combined with DNA inOptimem and incubated and added to cells according to manufacturer'sinstructions.

Transfections for H4 cells: 6 μL Fugene/well. Combined with DNA inOptimem and incubated and added to cells according to manufacturer'sinstructions.

Added 10 μM L-685,458 to Plates 1 and 2, wells 2 and 4 for both celltypes within 1 hour of transfection. Observed cells periodically.

Took pictures at 24, 46 hours after transfection, using AE lock to keepexposures constant between wells.

Results:

-   Both H4 and 293T cells turned much brighter green in the presence of    pcDNA3.1 zeo (+) APP(1-65 1)SW, K612V-(M1L)TATexonI    At 24 hours:    H4 cells:    Plate 1: 1 μg pMM321

1. 1 ug pMM321+1 ug pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI:Many bright and dim green cells (good transfection efficiency as well)

2. 1 ug pMM321+1 ug pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI+10 μM L-685,458: Also many bright and dim greencells, but reduced compared with well #1

3. Very few green cells (a few per field)

4. Very few green cells

5. A few dimly green cells

6. Some induction with 0.1 μg pUCd5TAT but still relatively few cells.

Plate 2: 0.1 μg pMM321

1. 0.1 μg pMM321+1 μg pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI: ˜10 bright green cells/field and the rest are dimgreen

2. 0.1 μg pMM321+1 μg pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI+10 μM L-685,458: ˜3-5 bright green cells/field, somedim green, and some not green.

3. No green cells

4. No green cells

5. No green cells

6. A few bright green cells

HEK293T Cells:

Plate 1: 1μg pMM321

1. +1 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI: of 15cells: 6 dim, 4 medium, 5 bright

2. +1 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI+10 μML-685,458: of 15 cells: 6 very dim, 5 dim, 1 medium, 3 bright

3. +0.1 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI: many moregreen cells than 1 μg, lots of strong, bright green cells

4. +0.1 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI+10 μML-685,458: Fewer bright green cells/field but intensity does not appearstrongly diminished

5. 1 μg pMM321 alone: Most cells in the field expressing dim to mediumlevels of GFP

6. enhancement by 0.1 μg pUCd5TAT

Plate 2: 0.1 μg pMM321

1. +1 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI: Bright andmedium green cells

2. +1 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI+10 μML-685,458: Bright, medium, and dim green cells

3. +0.1 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(1L)TATexonI: Bright,medium, and dim green cells

4. +0.1 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(41L)TATexonI+10 μML-685,458: Bright, medium, and dim green cells

5. 0.1 μg pMM321 alone: Most expressing cells have dim GFP, a few mediumto bright cells

6. Enhancement by 0.1 μg pUCd5TAT

Changed media on cells at 24 hours past transfection. Kept 10 μML-685,458 on cells in wells 2 and 4.

At 46 hours after transfection, examined the wells again. Lots offloating cells in all wells, all cell types. Highest number of floatersin 1 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI lanes.

Took some photographs under fluorescent and white light (white light atlow intensity) to reveal fluorescent and non-fluorescent cells.Conducted a subjective analysis of the photographs to see if the amountof inhibition by 10 μL-685,458 was in any way quantifiable. Countedbright (white in the middle), strong (blue middle), medium (green) anddim/non-fluorescent cells and determined the approximate fraction ofeach level of expression. Results follow: TABLE 1 293T cells transfectedwith X μg pMM321 (first number in left column) and X μg pcDNA3.1 zeo (+)APP(1-651)SW, K612V-(M1L)TATexonI (second number in left column)Transfection # Bright # Strong # Med # Non % Bright % Strong % Med % Non1 μg + 1 μg none 8 81 316 2 20 78 1 + 1 + cmpd none 5 66 521 0.8 11 881 + 0.1 41 77 143 61 12 24 44 19 1 + 0.1 + cmpd 23 32 149 194

The results shown in Table 1 indicate that the presence of L-685,458(“cmpd”) caused fewer strong and medium fluorescing cells as well asmore non-fluorescent cells in the first run; in the second run,L-685,458 caused fewer bright and strong fluorescing cells as well asmore non-fluorescent cells (with slightly more medium fluorescingcells). Overall, these data clearly indicate that the presence of aninhibitor of APP processing such as L-685,458 can be identified by thepresent invention.

1 mL of conditioned media from each well was analyzed for production ofAβ. Higher than background levels of Aβ were observed in 293T cellsafter transfection with 1 μg pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI and 0.1 μg pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI and higher than background levels of Aβ in H4 cellsafter transfection with 1 μg pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI, but not 0.1 μg pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI (no enhancement of GFP was observed with 0.1 μgpcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI either). Aβ wascompletely inhibited to background levels by 10 μM L-685,458.Surprisingly, substantial inhibition of GFP was not observed with 10 μML-685,458.

100,000 cells from each well were trypsinized and placed in 0.1 mLphenol red-free media in a Costar 96-well dish and read using thefluorometer. The results are shown below: TABLE 2 1 ug 10 uM 0.1 ug 10uM 0.1 ug APPTAT 458 APPtat 458 no tat pUCd5TAT 293T   1 ug LTRGFP 1767014321 65535 65535 14890 65535 0.1 ug LTRGFP 9976 10491 17677 14790 962425735 H4   1 ug LTRGFP 21307 25307 7175 7136 7147 7277 0.1 ug LTRGFP9574 10031 7317 6957 6946 7247 Blank 7498 7124 7570 7454 5774 7638

In Table 2, “APPtat” is pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI. “458” is L-685,458. “LTRGFP” is pMM321.

0.1 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI with andwithout compound exceeded the maximum reading of the fluorometer, as didthe addition of 0.1 μg pUCd5TAT to cells transfected with 1 μg pMM321.

1 μg pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI+1 μg pMM321incrementally increased the amount of fluorescence relative to 1 μgpMM321 alone, and this was reduced to background levels by 10 μML-685,458. Inhibition of fluorescence was also observed in 293T cellstransfected with 0.1 μg pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI+0.1 μg pMM321. No inhibition of fluorescence wasobserved in H4 cells under any transfection conditions.

EXAMPLE 3 Use of APP(1-651)SW, K612V-TATexonI in H4 Cells

L-875,532 is a known γ-secretase inhibitor having the structure shownbelow. It is described and details of its synthesis are disclosed inSeiffert et al., 2000, J. Biol. Chem. 275:34086-34091.

Compound X is a β-secretase inhibitor.

pRBR186 (FIG. 22A) is an expression vector containing DNA sequencesencoding full-length APP containing the Swedish mutation and the K612Vmutation. pRBR186 does not contain a transcription factor fused to theAPP sequences.

pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI is an expressionvector that directs the expression of a the fusion protein APP(1-651)SW,K612V-(M1L)TATexonI in mammalian cells. This fusion protein contains thefirst 651 amino acids of APP (with a Swedish version of the β-secretasecleavage site as well as the K612V mutation) fused in frame to exon I ofHIV1 TAT, which has been modified with a Met1-Leu mutation. A schematicdiagram of pcDNA3.1 zeo (+) APP(1-651)SW, K612V-(M1L)TATexonI is shownin FIG. 26A. The nucleotide sequence of pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI (SEQ ID NO:22) is shown in FIG. 26B-G.

Methods:

1. H4 cells (ATCC HTB-148) were transfected with the various constructslisted below using 6 μL Fugene per 100 μL Optimem and 100 μL Optimem perwell (6-well dishes). Transfection reactions were incubated for 30minutes prior to adding 100 μL dropwise onto wells.

Transfections were done as follows:

1. 1 μg pMM321 (FIG. 25A-D) and 1 μg pcDNA3.1 backbone

1a. 1 μg pMM321 and 1 μg pcDNA3.1 (Invitrogen, San Diego, Calif.)backbone. Prior to transfection, 10 μM L-875,532 (γ-secretase inhibitor)was added to the well.

2. 1 μg pMM321 and 1 μg pRBR186 (FIG. 22A; APP expression vector;processing and inhibition of processing control)

2a. 1 μg pMM321 and 1 μg pRBR186. Prior to transfection, 10 μM L-875,532was added to cells (transfection solution for 3-5 were prepared in bulk)

3. and 3a. 1 μg pMM321 and 1 μg pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI

4. and 4a. 1 μg pMM321 and 1 μg pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI. Prior to transfection, 10 μM L-875,532 was added tothe two wells.

5. and 5a. 1 μg pMM321 and 1 μg pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI. Prior to transfection, 10 μM Compound X was addedto the two wells

6. 1 μg pMM321 and 1 μg pUCd5TAT (FIG. 24).

7. 1 μg pMM321 and 1 μg pUCd5 TAT. Prior to transfection, 10 μML-875,532 was added to the cells.

Results:

Cells were assessed by eye under a fluorescence microscope the morningfollowing transfection (˜20 hrs).

1 and 1a, 2 and 2a. Weak fluorescence

3 and 3a. Much stronger fluorescence

4 and 4a. Clear inhibition of fluorescence

5 and 5a. Possible inhibition of fluorescence, but doesn't look thatgreat

6 and 7. Almost blindingly fluorescent.

At approximately 48 hours, cells were trypsinized, spun down, andresuspended in 100 μL PBS. The cellular contents of each well of thetransfection plates were placed into one well of a 96-well fluorescenceplate. Fluorescence was analyzed using the FLUOstar (485 excitation/538emission). The results are shown in Table 3. TABLE 3 Transienttransfections in H4 cells Fluor Units pMM321 4484 pMM321 + L-875,5323443 pMM321 + pRBR186 2735 pMM321 + pRBR186 + L-875,532 2161 pMM321 +APP-TAT-ct32 20177 pMM321 + APP-TAT-ct32 + L-875,532 8283 pMM321 +APP-TAT-ct32 + Compound X 11946 pMM321 + pucd5-TAT 61102

In Table 3, “APP-TAT-ct32” refers to pcDNA3.1 zeo (+) APP(1-651)SW,K612V-(M1L)TATexonI.

For a graphical presentation of these results, see FIG. 19. In FIG. 19,“LTR-GFP” refers to pMM321; “APP-TAT-ct32” refers to pcDNA3.1 zeo (+)APP(1-651)SW, K612V-(M1L)TATexonI. Compare the bar labeled“LTR-GFP+APP-TAT-ct32” with the bars labeled“LTR-GFP+APP-TAT-ct32+L-875,532” and “LTR-GFP+APP-TAT-ct32+Compound X.”Inhibition by both the β-secretase inhibitor (Compound X) and theγ-secretase inhibitor (L-875,532) is easily identified by the presentinvention.

CONCLUSIONS

The data indicate that the expression of the fusion proteinAPP(1-651)SW, K612V-(M1L)TATexonI enhances transactivation through theLTR of pMM321 in a manner that depends on APP processing.

APP(1-651)SW, K612V-(M1L)TATexonI expressing cells were 6× brighter thanpMM321 cells alone.

Treatment with L-875,532 decreased fluorescence 2.5×.

Treatment with Compound X decreased fluorescence 1.7×.

Expression of TAT via pucd5-TAT was almost blinding and was 19× abovepMM321 alone, indicating that APP(1-651)SW, K612V-(M1L)TATexonIexpression did not lead to levels of GFP as high as TAT alone. Despitethe decreased activation shown by TAT when provided by the fusionprotein, as compared with TAT driven by the AMLP (adenovirus major latepromoter) in pucd5-TAT, the assay was easily able to identify both theβ-secretase and the γ-secretase inhibitors.

Control plasmids (pMM321 and pMM321 +pRBR186) were dimly fluorescent andwere not inhibited by L-875,532.

A lower level of inhibition by the β-secretase inhibitor is to beexpected since the K612V mutation decreases alpha-secretase activity by95% and thus some alpha-secretase cleavage is to be expected.

EXAMPLE 4 Construction of pcDNA3.1 (+) zeo APP(1-651)SW, K612V,GAL4-VP16(M1L) APP (664-695)

1. The GAL4-VP16 insert was prepared by PCR from pCR2.1 GAL4VP16 (FIG.20) (Invitrogen, San Diego, Calif.). The PCR was performed to eliminatethe N-terminal methionine by changing this methionine into a leucine.

-   40 ng pCR2.1 GAL4-VP16

0.2 μL GAL4-VP16 5′ oligo at 250 μM: (SEQ ID NO:56)5′-CTGAGATATCAAGCTACTGTCTTCTATCGAACAAGC-3′

-   EcoRV site underlined

0.2 μL GAL4VP16 3′ oligo (at 250 μM): (SEQ ID NO:57)5′-GCGCGATATCCCCACCGTACTCGTCAATTCC-3′

-   EcoRV site underlined-   5 μL 10× Buffer-   8 μL 25 mM MgCl₂-   4 μL PCR dNTPs-   0.25 μL AmpliTaq Gold-   27.35 μL water    Cycle:-   Purified reactions using a Qiaquick column-   Digested entire reaction using EcoRV-   Ran the DNA on a 1% gel. Excised the band and purified using a    QiaQuick gel purification kit

2. Digested pcDNA3.1 APP(1-651)/APP(664-695) with EcoRV and SAP treated.pcDNA3.1 APP(1-651)/APP(664-695) is an intermediate plasmid formed inthe procedure described in Example 6. pcDNA3.1 APP(1-651)/APP(664-695)the first 651 amino acids of APP (with a Swedish version of theβ-secretase cleavage site as well as the K612V mutation) fused in frameto the last 32 amino acids of APP.

3. Ligated pcDNA3.1 APP(1-651)/APP(664-695) -EcoRV digested to GAL4VP16(EcoRV digested)

4. At this point, it was realized that the 3′ PCR primer for GAL4-VP16put the APP(664-695) fragment out of frame. The APP(664-695) fragmentwas then re-PCR'd using the following protocol:

-   1 μL pcDNA3.1 APP(1-651)-Gal4VP16-APP(664-695)-   50 nM APP NotI 5′ ct32 in frame with GAL4-VP16

50 nM APP Noti 3′ ct32 (SEQ ID NO:58)5′(p)CTGCTGTGGCGGCCGCCTAGTTCTGCATCTGCTC)

-   NotI site underlined-   1 μL PCR dNTPs (10 mM each dNTP, Roche)-   5 μL 10× Expand Buffer with MgCl₂-   40 μL water-   1 μL Expand polymerase (Roche)

The PCR fragment was run on a 4% agarose gel and gel-purified using aQiaQuick gel purification column

The fragment was digested with NotI and purified using a QiaQuick PCRpurification column.

5. APP(1-651)-Gal4VP16-APP(664-695) was re-miniprepped. Miniprep #1 wasdigested with NotI, run on a 1% gel, the upper band was then isolatedand SAP-treated.

6. APP(1-651)-Gal4VP16/NotI digested/SAP-treated was ligated toAPP(664-695).

7. Minipreps containing inserts were sequenced to verify the orientationof the insert.

EXAMPLE 5 Construction of pcDNA3.1 zeo (+) APP(1-651)wt,K612V-TATexonI(M1L) APP(664-695)

This procedure replaced a fragment of APP in pcDNA3.1 zeo (+)APP(1-651)SW, K612V-TATexonI(N41L) APP (664-695) that contained theSwedish mutation with a corresponding fragment from pRBR121 containingthe wild-type β-secretase cleavage site rather than the Swedishβ-secretase cleavage site.

1. pcDNA3.1 zeo (+) APP(1-651)SW, K612V-TATexonI(M1L) APP (664-695)(FIG. 21B) was digested with SnaBI and then EcoRI

17 μL miniprep DNA

2 μL 10× buffer

1 μL SnaBI (NEB)

Digest was purified using Qiaquick PCR purification kit. Entire digestwas then cleaved with EcoRI for 2 hours.

2. pRBR121 (FIG. 21A) was digested with SnaBI and then EcoRI

5 μg pRBR121

5 μL 10× buffer

2.5 μL SnaBI (NEB)

q.s. 50 μL with water

The digest was purified using a Qiaquick PCR purification kit. Theentire digest was then cleaved with EcoRI for 2 hours.

3. Both digests were run out on a 1% agarose gel. From the pRBR121 lane,the 2.4 kb SnaBI-EcoRI fragment containing the wild-type β-secretasecleavage site was isolated.

From pcDNA3.1 zeo (+) APP(1-651)SW, K612V-TATexonI(MIL) APP (664-695)digest, BOTH the 5 kb SnaBI-EcoRI backbone fragment AND the 200 bpEcoRI-EcoRI fragment were isolated (see FIG. 21B).

4. A three-part ligation using equal molar ratios of the three fragmentswas carried out:

The assumption was made that, since the starting plasmids were ofsimilar sizes and the same amount was digested for each plasmid, therecovered fragments would be in approximately equal molar ratios.

Vector alone:

-   1μL APP-TAT-ct32 SnaBI/EcoRI 5Kb fragment-   7 μL water-   2 μL 5× buffer-   10 μL 2× buffer (Roche rapid ligation kit)-   1 μL T4 ligase    1+1+1-   1 μL pcDNA3.1 zeo (+) APP(1-651)SW, K612V-TATexonI(M1L) APP    (664-695) SnaBI/EcoRI 5KB fragment-   1 μL pRBR121 SnaBI/EcoRI 2.4 Kb insert-   1 μL pcDNA3.1 zeo (+) APP(1-651)SW, K612V-TATexonI(MIL) APP    (664-695) EcoRI/EcoRI 200 bp insert-   5 μL water-   2 μL 5× buffer-   10μL 2× buffer-   1 μL T4 ligase    1+1+ . . . 1 (in this 3-pt ligation, the ligation of two of the    fragments together was done 1^(st), then the third fragment was    added)-   1 μL pcDNA3.1 zeo (+) APP(1-651)SW, K612V-TATexonI(M1L) APP    (664-695) SnaBI/EcoRI 5Kb backbone-   1 μL pcDNA3.1 zeo (+) APP(1-651)SW, K612V-TATexonI(M1L) APP    (664-695) EcoRI/EcoRI 200 bp insert-   5 μL water-   2 μL 5× buffer-   10 μL 2× buffer-   1 μL T4 ligase-   waited 5 minutes-   then added 1 μL pRBR121 SnaBI/EcoRI 2.4 kb insert    1+1+3-   1 μL pcDNA3.1 zeo (+) APP(1-651)SW, K612V-TATexonI(M1L) APP    (664-695) SnaBI/EcoRI 5 kb backbone-   1 μL pRBR121 SnaBI/EcoRI 2.4 kb insert-   3 μL pcDNA3.1 zeo (+) APP(1-651)SW, K612V-TATexonI(MIL) APP    (664-695) EcoRI/EcoRI 200 bp insert-   3 μL water-   2 μL 5× buffer-   10 μL 2× buffer-   1 μL T4 ligase

Transformed and plated out 200 μL. The number of colonies in thevector+insert ligations far exceeded the number of colonies in thevector alone ligation. Picked 12 colonies from 1+1+ . . . 1.

-   Picked 6 colonies from 1+3.-   Miniprepped-   Digested with EcoRI to ensure that small 200 bp fragment was    incorporated.    Results:

Minipreps #10 and 15 contained 200 bp EcoRI fragment.

Oriented with Bam HI digestion.

Sequenced with sAPPb F2 and F3 primers. Miniprep #15 contains bothinserts in the correct orientation.

EXAMPLE 6 Construction of pcDNA3.1 zeo (+) APP(1-651)SW,K612V-TATexonI(M1L) APP(664-695)

1. PCR of APP (664-695):

-   The starting material was the pRBR186 plasmid (FIG. 22A).    PCR of APP (664-695)-   1 mg pRBR186

50 nM 5′ oligo (SEQ ID NO:59) (5′-TGCCCCGCGCGGCCGCGCGATGCTGCCCGG-3′)NotI site underlined

50 nM 3′ oligo (SEQ ID NO:60)(5′-(p)ATGGTGTGGCGGCCGCAGACGCCGCTGTCACC-3′)NotI site underlined

-   1 μL Roche PCR nucleotides-   5 μL 10× Expand buffer-   40 μL water-   1 μL Expand

Cycle: (94° C., 5 min)−25×(94° C., 30 sec; 42° C., 1 min; 72° C., 2min)−72° C. ×6 min−4° C. hold.

The ˜100 bp fragment was gel purified (4% agarose, 1×TBE gel)

The gel-purified fragment was ligated into NotI digested, ShrimpAlkaline Phosphatase-treated pcDNA3.1 zeo (+) (Invitrogen). The presenceof the insert and its orientation was confirmed by sequencing.

2. PCR of APP(1-651):

-   1 ng pRBR186

50 nM 5′ oligo (SEQ ID NO:61)(5′-(p)AGCGCACAAGCTTCCCCGCGCAGGGTCGCGATGCTG-3′)HindIII site underlined, Met(1) ATG of APP in bold

50 nM 3′ oligo (SEQ ID NO:62)(5′-(p)GGATGTAAGCTTTTTCTTCTTCAGCATCACCAAGG-3′)HindIII site underlined

-   1 μL Roche PCR nucleotides-   5 μL 10× Expand buffer-   40 μL water-   1 μL Expand

Cycle: (94° C., 5 min)−25×(94° C., 30 sec; 37° C., 1 min; 72° C. 2.5min)−72° C.×6 min−4° C. hold

The amplified fragment was isolated on an agarose gel. The fragment waspurified from the gel using Qiaquick Gel purification columns. Thefragment was digested with HindIII. The amount of the fragment was toosmall to subclone, so the PCR was repeated using 1 μL of the amplifiedfragment and carrying out 5 reactions simultaneously.

The fragments were purified from these reactions using a QiaQuick PCRpurification kit. The fragments were eluted in 30 μL and digested withHindIII for 2 hours. The digested fragments were then gel purified.

The purified fragments were ligated to pcDNA3.1 zeo (+) APP(664-695)that had been digested with HindIII and SAP treated. This gave theintermediate plasmid pcDNA3.1 zeo (+) APP(1-651)/APP(664-695).

3. PCR of (M1L) TAT:

The starting material was NL4-3 viral plasmid (FIG. 22B).

PCR reaction:

-   1 ng NL4-3 viral plasmid

50 nM TAT 5′ RV Met-Leu PCR primer (SEQ ID NO:63)(5′-TGCAGATATCCTGGAGCCAGTAGATCCTAGAC-3′)

-   EcoRV site underlined, Met-Leu mutation in bold

50 nM TAT 3′ RV Met-Leu PCR primer (SEQ ID NO:64)(5′-GCTGGATATCCTCTGCTTTGATAGAGAAGC-3′)

-   EcoRV site underlined-   1 μL PCR dNTPs-   5 μL PCR 10× buffer with MgCl₂-   40 μL water-   1 μL Expand polymerase    Cycle:-   94° C. for 5 min-   [30 sec 94° C., 1 min 42° C., 1 min 72° C.]×25 cycles-   5 min at 72C-   hold at 4° C.

The insert was purified over QiaQuick PCR purification column

The entire reaction was digested with 30 units EcoRV for 3 hours

The ˜200 bp insert was gel purified.

pcDNA3.1 zeo (+) APP(1-651)/APP(664-695) was digested with EcoRV, andthen SAP treated

The Met1-Leu TAT fragment was ligated to pcDNA3.1 zeo (+)APP(1-651)/APP(664-695).

A map of the resulting plasmid is shown in FIG. 22C.

EXAMPLE 7 Design of Novel Expression Vector for Expression ofAPP(1-651)SW, K612V-TATexonI(M1L) APP(664-695)

Purpose:

To provide a low level expression of APP(1-651)SW, K612V-TATexonI(M1L)APP(664-695), a prokaryotic selectable marker that is NOT ampicillin(read-through of the β-lactamase gene is sometimes a problem), and aeukaryotic selectable marker that is NOT zeocin (zeo is the marker forthe reporter plasmids in some embodiments).

Methods

1. The dEYFP gene was removed from pd2EYFP (Clontech, Palo Alto, Calif.)using BamHI and NotI. The 5′ overhangs was filled in using Klenow, andthe plasmid was re-circularized.

-   pd2EYFP plasmid was digested with BamHI, NotI.-   Ran reaction on 1% agarose gel. Digestion was complete. Cut out 3.4    kb band.-   Purified using Qiagen Gel Extraction Kit.    Klenow Fill-In:-   ˜4 μg plasmid backbone-   7.5 μL NEB buffer 2-   33 μM each dNTP (diluted from Roche PCR dNTPs)-   water to 75 μL-   4 μL Roche Klenow fragment (4 units)-   Incubated at room temperature for 15 minutes-   Heat inactivated at 70° C.-   Took 1 μL fill-in reaction.-   Diluted to 8 μL with water-   Added 2 μL 5×DNA buffer-   Added 10 μL 2× Ligation Buffer-   Added 1 μL T4 DNA ligase-   Incubated at room temperature

Transformed 2 μL ligation into Invitrogen maximum efficiency DH5alphacompetent cells.

Plated out on Kanamycin plates. Lots of colonies.

2. The RSV promoter from pREP4 (Invitrogen) was excised using BglII andHindIII and cloned into the re-circularized plasmid.

-   Digested 5 μg pREP4 with HindIII-   Purified using Qiagen PCR purification kit-   Digested with BglII.

The RSV promoter fragment was gel purified and cloned into there-circularized plasmid.

3. The resulting expression vector (pRSV Kan/Neo res; FIG. 23) has theeukaryotic RSV promoter 5′ to the pd2EYFP polylinker, SV40 driving neoand kanamycin prokaryotic selection, and a pUC ori for high levels ofreplication in bacteria.

EXAMPLE 8 Use of APP(1-651)SW, K612V-TATexonI(M1L) APP(664-695) in HeLaCells With a β-galactosidase Reporter Gene

The following demonstrates the practice of the present invention withthe APP(1-651)SW, K612V-TATexonI(M1L) APP(664-695) fusion protein (SEQID NO:2) and β-galactosidase as a reporter gene. P4-R5 cells are HeLacells that contain a stably integrated β-galactosidase reporter geneunder the control of the HIV1 LTR.

Materials:

1.) Cells: P4-R5 cells

2.) DNA: 0.78 μg/μL pcDNA3.1 zeo (+)APP(1-651)SW, K612V-TATexonI(M1L)APP(664-695)

3.) Transfection reagents: FUGENE®

4.) Media: OPTIMEM®

-   cDMEM (−)phenol red/10% FBS

5.) Compounds: Compound X (β-secretase inhibitor) 10 mM

-   L-875,532 (γ-secretase inhibitor) 10 mM    Protocol:    Day 1

1.) Cell count on P4-R5 cells=7.6×10⁵ cells per ml in cDMEM (−)PR.Seeded sterile white luminometer TC plates with the following cellnumbers:

10 ml

-   5×10³/well=0.75 ml in 9.25 ml media-   1.0×10⁴/well=1.5 ml in 8.5 ml media-   Seeded 100 μL cells per well.-   Incubate overnight at 37° C., 5% CO₂.    Day 2

2.) Made up media with appropriate dilutions of inhibitors.

-   On no-inhibitor controls, added 100 μL of cDMEM with 1% DMSO-   On wells with Compound X, added 10 μM inhibitor in cDMEM-   On wells with L-875,532, added 10 μM inhibitor in cDMEM

3.) Prior to transfection, pulled off media on P4-R5 cells and replacedwith media −/+inhibitor.

-   FUGENE® transfection:-   For FUGENE® transfection:

4.) Added 600 μL of OPTIMEM® to sterile EPPENDORF® tube and carefullyadded 30 μL FUGENE® to media, without touching walls of tube. Incubatedat room temperature for 5 minutes.

-   In separate EPPENDORF® tubes, added each DNA.-   Added FUGENE®/OPTIMEM® dropwise to DNA; incubated at room    temperature for 15 minutes.

Added 15 μL/well of DNA/FUGENE®/OPTIMEM® dropwise to media inappropriate wells on P4-R5 cells, swirling to mix. TABLE 4 Transfectionnumber Conc of DNA Vol of DNA Vol of FUGENE ® Vol of sterile OPTIMEM ®APP-ML-Tat-APPct 0.78 μg/μL 5 μg = 6.5 μL 30 μL of FUGENE ® 600 μL ofOPTIMEM ® pUCd5TAT 1.24 μg/μL 5 μg = 4.0 μL 30 μL of FUGENE ® 600 μL ofOPTIMEM ® p243-4 0.56 μg/μL 5 μg = 9 μL 30 μL of FUGENE ® 600 μL ofOPTIMEM ®

In Table 4, “APP-ML-Tat-APPct” refers to pcDNA3.1 zeo (+)APP(1-651)SW,K612V-TATexonI(M1L) APP(664-695). “pUCd5TAT” is an expression vectorthat serves as a positive control for TAT expression, since it is aconstruct in which TAT is under the control of a strong, constitutivepromoter (see FIG. 24). “p243-4” is a control expression vector thatdirects the expression of APP.

5.) Plates were transferred to an incubator and incubated for 48 hoursto allow expression and processing of the proteins.

Day 4

6.) The protocol below was followed for lysis of the cells andmeasurement of β-galactosidase in the cell lysates.

Measurement of β-galactosidase in lysates of transfected cells.

1. Removed TROPIX® chemiluminescence kit(s) from cold room, allowed tocome to room temperature in a 37° C. water bath.

2. β-galactosidase standards were prepared: Made 1:5000 dilution ofβ-galactosidase stock (1 mg/ml) in lysis buffer. Did 2 fold dilutions.

3. Diluted TROPIX® substrate 1:25 into buffer. (Made enough for 100μL/well).

4. Added to reservoir and added 100 μL/well.

5. Added 10 μL of β-galactosidase standards to column 12 on plate andincubated in dark for 1 hour.

6. Read immediately in luminometer using standard file. Filled inrequired fields, read plate.

The results are shown in FIG. 33. FIG. 33 demonstrates that the presentinvention was able to identify both the β-secretase inhibitor (CompoundX) and the γ-secretase inhibitor (L-875,532). In FIG. 33, “APP-tat-ct32”refers to pcDNA3.1 zeo (+)APP(1-651)SW, K612V-TATexonI(M1L)APP(664-695). Although not indicated in FIG. 33, the results for thecontrols were as expected: a large transactivation of the LTR bypUCd5TAT was observed which was not affected by either inhibitor. Notransactivation was seen with p243-4.

EXAMPLE 9 Comparison of the Use of APP(1-651)SW, K612V-TATexonI(M1L)APP(664-695) and APP(1-651)wt, K612V-TATexonI(M1L) APP(664-695) With aβ-galactosidase Reporter Gene

The following shows a side-by-side comparison of the practice of thepresent invention with the APP(1-651)SW, K612V-TATexonI(M1L)APP(664-695) fusion protein (SEQ ID NO:2) and the APP(1-651)wt,K612V-TATexonI(M1L) APP(664-695) fusion protein (SEQ ID NO:4). P4-R5cells are HeLa cells that contain a stably integrated β-galactosidasereporter gene under the control of the HIV1 LTR.

Materials:

1.) Cells: P4-R5 cells

2.) DNA: 0.78 μg/μL pcDNA3.1 neo (+) APP(1-651)SW, K612V-TATexonI(M1L)APP(664-695)

-   0.812 μg/μL pcDNA3.1 neo (+) APP(1-651)wt, K612V-TATexonI(M1L)    APP(664-695)-   1.24 μg/μL pUCd5TAT-   0.56 μg/μL p243-4

3.) Transfection reagents: FUGENE®

4.) Media: OPTIMEM®

-   cDMEM (−)phenol red/10% FBS

5.) Compounds: Compound X (β-secretase inhibitor) 10 mM

-   L-875,532 (γ-secretase inhibitor) 10 mM    Day 1

1.) Cell count on P4-R5 cells=5×10⁵ cells per ml in cDMEM (−)PR. Seededsterile white luminometer TC plates with the following cell numbers:

10 ml

-   5×10³/well=4.0 ml in 36.0 ml media-   Diluted 1:1 into media and seeded one plate at 2.5×10³/well.-   Seeded 100 μL cells per well.-   Incubated overnight at 37° C., 5% CO₂.    Day 2

2.) Made up media with appropriate dilutions of inhibitors.

-   On no-inhibitor controls, added 100 μL of cDMEM with 1% DMSO-   On wells with Compound X, added titration curve from 100 μM    inhibitor in cDMEM (−)PR.-   On wells with L-875,532, added titration curve from 100 μM inhibitor    in cDMEM (−)PR.

3.) Prior to transfection, pulled off media on P4-R5 cells and replacedwith media −/+ inhibitor.

FUGENE® Transfection:

4.) Added volume of OPTIMEM® to sterile EPPENDORF® tube and carefullyadded correct volume of FUGENE® to media, without touching walls oftube. Incubated at room temperature for 5 minutes.

-   In separate EPPENDORF® tubes, added each DNA, as outlined below.-   Added FUGENE®/OPTIMEM® dropwise to DNA; incubated at room    temperature for 15 minutes.

Added 15 μL/well of DNA/FUGENE®/OPTIMEM® dropwise to media inappropriate wells on P4-R5 cells, swirling to mix. TABLE 5 Transfectionnumber Conc of DNA Vol of DNA Vol of FUGENE ® Vol of sterile OPTIMEM ®1.) APP-ML-Tat-APPct (Sw) 0.78 μg/μL 10 μg = 13 μL 60 μL of FUGENE ®1200 μL of OPTIMEM ® 2.) APP-ML-Tat-APPct (WT) 0.812 μg/μL  10 μg = 12.2μL 60 μL of FUGENE ® 1200 μL of OPTIMEM ® 3.) pUCd5TAT 1.24 μg/μL  5 μg= 4.0 μL 30 μL of FUGENE ®  600 μL of OPTIMEM ® 4.) p243-4 0.56 μg/μL  5μg = 9 μL 30 μL of FUGENE ®  600 μL of OPTIMEM ®

In Table 5, “APP-ML-Tat-APPct (Sw)” refers to pcDNA3.1 neo (+)APP(1-651)SW, K612V-TATexonI(M1L) APP(664-695). “APP-ML-Tat-APPct (WT)”refers to pcDNA3.1 neo (+) APP(1-651)wt, K612V-TATexonI(M1L)APP(664-695). “pUCd5TAT” is an expression vector that serves as apositive control for TAT expression, since it is a construct in whichTAT is under the control of a strong, constitutive promoter (see FIG.24). “p243-4” is a control expression vector that directs the expressionof APP.

5.) Plates were transferred to an incubator and incubated for 36 hoursto allow expression and processing of proteins.

Day 4

6.) The protocol below was followed for lysis of the cells andmeasurement of β-galactosidase in the cell lysates.

Measurement of β-galactosidase in Lysates of Transfected Cells.

1. Removed TROPIX® chemiluminescence kit(s) from cold room, allowed tocome to room temperature in a 37° C. water bath.

2. β-galactosidase standards were prepared: Made 1:5000 dilution ofβ-galactosidase stock (1 mg/ml) in lysis buffer. Did 2 fold dilutions.

3. Diluted TROPIX® substrate 1:25 into buffer. (Made enough for 120μL/well).

4. Added to reservoir and added 120 μL/well.

5. Added 10 μL of β-galactosidase standards to column 12 on plate andincubated in dark for 1 hour.

6. Read immediately in luminometer using standard file. Filled inrequired fields, read plate.

The results are shown in FIG. 34. In FIG. 34, “APP(NFEV)HAMycFLAG”refers to a protein that is a variant of APP in which NFEV is present atthe β-secretase cleavage site and there are epitope tags in the aminoterminal portion of the protein but there is no transcription factorfused to APP. “APP(Sw)tat-ct32” refers to pcDNA3.1 neo (+) APP(1-651)SW,K612V-TATexonI(M1L) APP(664-695). “APP(WT)tat-ct32” refers to pcDNA3.1neo (+) APP(1-651)wt, K612V-TATexonI(M1L) APP(664-695). FIG. 34 showsthat the Swedish version and the wild-type version of APP appear to workabout equally well in the assay.

EXAMPLE 10 L-685,458

L-685,458 is a γ-secretase inhibitor having the following structure:

L-685,458 contains an hydroxyethylene dipeptide isostere and is thoughtto function as a transition state analog mimic of aspartyl proteases(Shearman et al., 2000, Biochemistry 39:8698-8704). L-685,458 wasprepared as follows:{1S-Benzyl-4R-[1-(1S-carbamoyl-2-phenylethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydroxy-5-phenylpentyl}carbamicacid tert-butyl ester (L-685,458) was prepared by the coupling of2R-benzyl-5S-tert-butoxycarbonylamino-4R-(tert-butyldimethylsilanyloxy)-6-phenylhexanoicacid (Evans et al., 1985, J. Org. Chem. 50:4615-4625) with Leu-Phe-NH2followed by deprotection with tetrabutylammonium fluoride. The synthesisof{1S-benzyl-4R-[1-(1S-carbamoyl-2-phenylethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2S-hydroxy-5-phenylpentyl}carbamicacid tert-butyl ester (L-682,679) has been described previously (DeSolms et al., 1991, J. Med. Chem. 34:2852-2857).{1S-Benzyl-4R-[1-(1S-carbamoyl-2-phenylethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2-oxo-5-phenylpentyl}carbamicacid tert-butyl ester (L-684,414) was prepared by pyridiniumdichromate-mediated oxidation of L-682,679.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A DNA molecule comprising a nucleotide sequence encoding a fusionprotein comprising amino acids 589-651 selected from the groupconsisting of wild type APP₆₉₅, the Swedish version of APP₆₉₅ and theNFEV (SEQ ID NO:40) version of APP₆₉₅ and a transcription factor wherethe transcription factor is fused in frame to the carboxyl terminus ofamino acids 589-651.
 2. The DNA molecule of claim 1 where amino acids589-651 contain a K612V mutation.
 3. The DNA molecule of claim 1 wherethe nucleotide sequence further encodes amino acids 664-695 of APP₆₉₅wherein amino acids 664-695 are fused in frame to the carboxyl terminusof the transcription factor.
 4. The DNA molecule of claim 1 where thetranscription factor is selected from the group consisting of: HIV-1TAT, Gal4-VP16, the entire Gal4 protein, LexA-VP16, E12, E47, Twist,Papillomavirus E2, EBV Zta, BIV TAT, HIV-2 TAT, or SIV TAT.
 5. The DNAmolecule of claim 3 where the fusion protein is selected from the groupconsisting of: APP(1-651)wt, K612V-TATexonI(M1L) APP (664-695) (SEQ IDNO:4); APP(1-651)wt, K612V, Gal4-VP16(M1L) APP (664-695) (SEQ ID NO:8);APP(1-651)wt, TATexonI(M1L) APP (664-695) (SEQ ID NO:12); andAPP(1-651)wt, Gal4-VP16(M1L) APP (664-695) (SEQ ID NO:16).
 6. The DNAmolecule of claim 3 where the fusion protein is selected from the groupconsisting of: APP(1-651)SW, K612V-TATexonI(M1L) APP (664-695) (SEQ IDNO:2); APP(1-651)SW, K612V, Gal4-VP16(M1L) APP (664-695) (SEQ ID NO:6);APP(1-651)SW, TATexonI(M1L) APP (664-695) (SEQ ID NO:10); andAPP(1-651)SW, Gal4-VP16(M1L) APP (664-695) (SEQ ID NO:14).
 7. The DNAmolecule of claim 3 where the fusion protein is selected from the groupconsisting of: APP(1-651)NFEV, K612V-TATexonI(M1L) APP (664-695) (SEQ IDNO:23) and APP(1-651)NFEV, K612V, GAL4-VP16(M1L) APP (664-695) (SEQ IDNO:25).
 8. An expression vector comprising the DNA molecule of claim 1.9. A eukaryotic cell comprising the DNA molecule of claim
 1. 10. Thecell of claim 9 further comprising a reporter gene where the reportergene is under the control of a regulatory DNA sequence that is capableof being activated by the transcription factor.
 11. A method ofidentifying a substance that inhibits APP processing comprising: (a)providing a recombinant eukaryotic cell which: (i) expresses a fusionprotein comprising amino acids 589-651 selected from the groupconsisting of wild type APP₆₉₅, the Swedish version of APP₆₉₅ and theNFEV (SEQ ID NO:40) version of APP₆₉₅ and a transcription factor wherethe transcription factor is fused in frame to the carboxyl terminus ofamino acids 589-651; and (ii) comprises a reporter gene operably linkedto a regulatory DNA sequence which capable of being activated by thetranscription factor; (b) measuring the level of reporter gene productin the cell in the absence of the substance; (c) adding the compound tothe cell and measuring the level of reporter gene product in the cell inthe presence of the substance; where a decrease in the level of reportergene product in the presence as compared to the absence of the substanceindicates that the substance inhibits APP processing.
 12. The method ofclaim 11 where amino acids 589-651 contain a K612V mutation.
 13. Themethod of claim 11 where the transcription factor is selected from thegroup consisting of: HIV-1 TAT, Gal4-VP16, the entire Gal4 protein,LexA-VP16, E12, E47, Twist, Papillomavirus E2, EBV Zta, BIV TAT, HIV-2TAT, or SIV TAT.
 14. The method of claim 11 where the fusion protein isselected from the group consisting of: APP(1-651)wt, K612V-TATexonI(M1L)APP (664-695) (SEQ ID NO:4); APP(1-651)wt, K612V, Gal4-VP16(M1L) APP(664-695) (SEQ ID NO:8); APP(1-651)wt, TATexonI(M1L) APP (664-695) (SEQID NO:12); and APP(1-651)wt, Gal4-VP16(M1L) APP (664-695) (SEQ IDNO:16).
 15. The method of claim 11 where the fusion protein is selectedfrom the group consisting of: APP(1-651)SW, K612V-TATexonI(M1L) APP(664-695) (SEQ ID NO:2); APP(1-651)SW, K612V, Gal4-VP16(M1L) APP(664-695) (SEQ ID NO:6); APP(1-651)SW, TATexonI(M1L) APP (664-695) (SEQID NO:10); and APP(1-651)SW, Gal4-VP16(M1L) APP (664-695) (SEQ IDNO:14).
 16. The method of claim 11 where the fusion protein is selectedfrom the group consisting of: APP(1-651)NFEV, K612V-TATexonI(M1L) APP(664-695) (SEQ ID NO:23) and APP(1-651)NFEV, K612V, Gal4-VP16(M1L) APP(664-695) (SEQ ID NO:25).
 17. A method of identifying a substance thatinhibits APP processing comprising: (a) providing a recombinanteukaryotic cell which: (i) expresses a fusion protein comprising anamino acid sequence from APP that is capable of being cleaved by bothβ-secretase and γ-secretase and a transcription factor where thetranscription factor is fused in frame to the amino acid sequence fromAPP; and (ii) comprises a reporter gene operably linked to a regulatoryDNA sequence which capable of being activated by the transcriptionfactor; (b) measuring the level of reporter gene product in the cell inthe absence of the substance; (c) adding the compound to the cell andmeasuring the level of reporter gene product in the cell in the presenceof the substance; where a decrease in the level of reporter gene productin the presence as compared to the absence of the substance indicatesthat the substance inhibits APP processing.
 18. The method of claim 17where the amino acid sequence from APP comprises an amino acid sequenceselected from the group consisting of 589-651 of APP₆₉₅, 589-651 of theSwedish version of APP₆₉₅, and 589-651 of the NFEV version of APP₆₉₅.19. The method of claim 17 where the amino acid sequence from APPcontains the amino acid sequence NLDA (SEQ ID NO:38) at the β-secretasecleavage site instead of the wild-type sequence KMDA (SEQ ID NO:34). 20.The method of claim 17 where the amino acid sequence from APP containsthe amino acid sequence NFEV (SEQ ID NO:40) at the β-secretase cleavagesite instead of the wild-type sequence KMDA (SEQ ID NO:34).